STATUTORY DECLARATION

I hereby declare that I wrote the presented dissertation "Generation and characterization of a Dmd EGFP reporter mouse as a tool to investigate dystrophin expression" independently and used no other aids than those cited. In the written paragraphs, I have clearly identified the source of the passages that are quoted or paraphrased from other works. I also hereby declare that I have conducted my scientific work according to the principles of good scientific practice in accordance with the current "Richtlinien der Freien Universität Berlin" (Guidelines of the Free University of Berlin) and "Charte du doctorat à l'UPMC" (Charter of PhD students at UPMC). I, Mina Petkova, received assistance in the writing of this thesis with respect to grammar and syntax, which was provided by Markus Schuelke and Helge Amthor. I would like to express special thanks to all current and former co-workers of the Schuelke group in Berlin for their help, understanding, for their encouragment, and for the friendly and nice work atmosphere. I would especially like to thank Susanne, Esther and Franziska for their technical assistance. I am very thankful to Can for her unconditional support, great discussions, understanding and friendship. I am grateful to Evelyn and Sophie, students who helped and supported my project. I would also like to thank the members of the group of Prof.

Dr. H. Amthor and Dr. L. Garcia at the Université Saint-Quentin en Yvelines, especially to Amalia and Sonia.

I also would like to thank Josefine Radke and co-workers at the Institute of Neuropathology at the Charité Universitätsmedizin, Berlin for performing the histological analysis and western blots.

I would like to thank Polygene AG, Switzerland for the generation of the transgenic mice.

I am thankful to the group of Prof. Dr. Carmen Birchmeier from MDC-Berlin for providing the Cre-deleter mice.

I want to thank to all co-workers of the animal facilities of the Charité Cross Over building, as well as in the central animal facility FEM in Berlin for caring and breeding of experimental animals.

I would like to thank MyoGrad for giving me the opportunity to be part of a bi-national PhD program. Thanks to all students and to the organization team, especially Susanne Wissler.

I would like to thank my family, especially my parents and my grandmother for their love, for their support each step of the way, for giving me the chance to explore different opportunities, and ultimately to achieve this important goal.

Finally, I would like to thank all my friends for their patience, support, for believing in me and for the energy they gave me to accomplish my work.

The most beautiful thing we can experience is the mysterious.

It is the source of all true art and science. (Albert Einstein) 1. SUMMARY Dystrophin is a rod-shaped cytoplasmic protein that physically links the cytoskeleton to the ECM through the dystrophin-associated protein complex (DAPC), thereby providing sarcolemmal stability. Mutations in the dystrophin encoding DMD gene cause the severe Xlinked disorder Duchenne muscular dystrophy (DMD). DMD is characterized by progressive muscle wasting and fibrosis, impairing notably skeletal and heart muscle function as well as to various degrees cognitive, visual and gastrointestinal function due to missing dystrophin in the respective tissues. In this work a novel Dmd EGFP reporter mouse that expresses a fluorescently labelled endogenous dystrophin -EGFP fusion protein was generated and characterized. The protein was tagged at the C-terminus that is present in the most dystrophin isoforms. To date, no dystrophin reporter mice exist, thus imaging is only possible by indirect antibody-mediated processing ex vivo. For the generation of transgenic mice a targeting vector containing a FLAG-EGFP coding sequence inserted in-frame after the last modified exon 79 of the murine Dmd gene was constructed. Following the EGFP sequence a loxP flanked neomycin cassette was inserted into the 3'UTR. The vector was used for modification of the X-chromosomal Dmd locus in embryonic stem cells and germline transmission of the modified allele. After removal of the neomycin selection cassette in the F1 generation Dmd EGFP mice and their wildtype littermates were characterized.

Strong natural EGFP expression was observed in skeletal and smooth muscles, heart, brain and the eye and EGFP fluorescence co-localized with dystrophin at all sites suggesting proper tagging of the major dystrophin isoforms. In skeletal muscle, dystrophin as well as other proteins of the DAPC were expressed in normal quantity at correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting a correct function of the dystrophin-EGFP fusion protein. Isolated myofibers as well as satellite-cell derived myotubes expressed EGFP in vitro. Thus, the novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression.

Furthermore, the model can be used to investigate dystrophin re-expression in vivo or ex vivo after various gene therapy protocols that are aimed at the reestablishment of the dystrophin open reading frame or in naturally occurring revertant fibers.

ZUSAMMENFASSUNG

Dystrophin ist ein zytoplasmatisches

RÉSUMÉ

La dystrophine est une protéine cytoplasmique qui lie physiquement le cytosquelette à la matrice extracellulaire par le biais du complexe dystrophine-protéines associées (DAPC), assurant ainsi la stabilité du sarcolemme. Des mutations dans le gène DMD codant pour la dystrophine, conduisant à l'absence de la protéine, sont à l'origine de la dystrophie musculaire de Duchenne (DMD) qui est une maladie liée au chromosome X. La DMD est caractérisée par une atrophie et fibrose musculaire progressive, affectant les fonctions musculaires squelettiques et cardiaques. Les fonctions cognitives, visuelles et gastro-intestinales peuvent aussi être atteintes à différents niveaux. Pour mes travaux de thèse, j'ai généré et caractérisé un nouveau modèle de souris transgéniques rapportrices, dénommé Dmd EGFP , qui exprime une protéine dystrophine endogène fusionnée avec la protéine fluorescente EGFP. La protéine dystrophine est liée dans sa région C-terminale qui est présente dans la majorité des isoformes.

A ce jour, il n'existe aucune souris rapportrice pour la dystrophine et le marquage de la dystrophine doit se faire de manière indirecte par le biais d'anticorps. Pour la construction du vecteur de ciblage génique, la séquence codante pour FLAG-EGFP a été insérée après le dernier exon 79 du gène Dmd murin en respectant son cadre de lecture et dans lequel le codon de terminaison était enlevé. En aval de la séquence EGFP, une cassette ayant une séquence codante d'un gène de résistance à la néomycine, lui-même flanqué de sites loxP, était insérée afin de sélectionner les cellules ES. La recombinaison homologue dans le locus Dmd du chromosome X était vérifiée par la technique du Southern blot et les cellules ES étaient injectées dans des blastocystes. Après la transmission germinale, des animaux Dmd EGFP de la génération F1 étaient croisés avec des souris exprimant la Cre recombinase dans le but d'enlever la cassette néomycine. Le génotypage des souris était effectué par PCR.

Une expression forte et naturelle de l'EGFP était observée dans les muscles squelettiques, lisses, le coeur, le cerveau et l'oeil, ce qui suggère un étiquetage correct de tous les isoformes de la dystrophine. La fluorescence de l'EGFP co-localisait exactement avec la dystrophine dans tous les sites. Dans le muscle squelettique, la dystrophine ainsi que d'autres protéines de la DAPC étaient exprimées dans des quantités normales et dans la bonne localisation subsarcolemmale. De plus, l'architecture du tissu musculaire squelettique était normale, suggérant que la fonction de la protéine de fusion dystrophine-EGFP était maintenue. Par ailleurs, in vitro, l'EGFP est également exprimée dans les fibres musculaires isolées, ainsi que dans les myotubes dérivés des cellules satellites. Par conséquent, cette nouvelle souris rapportrice de la dystrophine devient un outil important pour la visualisation directe et in vivo de l'expression de la dystrophine.

De plus, le modèle peut être utilisé pour étudier la dystrophine ré-expression in vivo ou ex vivo après différents protocoles de thérapie génique qui visent à le rétablissement du cadre de lecture ouvert de la dystrophine ou en fibres révertantes d'origine naturelle.

INTRODUCTION

Dystrophin is large rod-like cytoskeletal protein that is primarily found at the inner surface of muscle fibers. It is expressed in all types of muscles as well as in the nervous system.

Dystrophin was identified as the protein which is absent in patients suffering from Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder. Most of our knowledge on the function of dystrophin is derived from studies on DMD patients as well from animal models of the disease. No cure for DMD has been found yet. Prevously unknown functions of dystrophin have been discovered recently leading the research on DMD into new directions. Hence there are still many questions to be answered in order to better understand the role of dystrophin in the normal and in the context of disease.

Pathology of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD; OMIM#310200) is the most common form of muscular dystrophies in childhood, a group of diseases characterized by progressive muscle degeneration, loss of the ability to walk, the impairment of repiratory and cardiac function and early death. Due to its transmission in families it was known that the disease is hereditary and X-linked, but the precise mechanism remained a mystery until 1986, when the causative genetic defect in DMD was discovered [START_REF] Koenig | Complete cloning of the duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF]A. P. Monaco et al., 1986) and its protein product, named dystrophin from then on, was identified [START_REF] Hoffman | Dystrophin: The protein product of the duchenne muscular dystrophy locus[END_REF]. It is either absent in or nearly absent DMD patients or truncated and reduced in the milder disease form, called Becker muscular dystrophy (BMD) [START_REF] Worton | Muscular dystrophies: diseases of the dystrophin-glycoprotein complex[END_REF].

Duchenne muscular dystrophy was first described by Guillaume-Benjamin-Amand Duchenne (de Boulogne) [START_REF] Duchenne | Recherches sur la paralysie musculaire pseudohypertrophique ou paralysie myo-sclérosique[END_REF] and William Richard Gowers (Gowers, 1878). It is the most common and severe form of an X-linked, recessive neuromuscular disorder, affecting 1 in 3,500 boys [START_REF] Emery | Abnormalities of the electrocardiogram in hereditary myopathies[END_REF]. Initial symptoms in DMD start in early childhood and include difficulties in running, in climbing stairs and a waddling gait. A characteristic sign of proximal muscle weakness is the so-called Gowers' phenomenon by which the child climbs up his thighs when trying to rise from the floor (Gowers, 1878). Typical features of the disease are the hypertrophy of the calf muscles, a progressive weakness and wasting of the proximal skeletal muscles (Figure 1). Ambulation is lost in most DMD patients by the age of 12 years and only a few patients survive until the third decade of life. Respiratory and/or cardiac failure are the main causes of death in DMD patients. Other organs being affected besides the skeletal muscles and myocardium are the smooth muscles as well as the central nervous system (CNS) [START_REF] Barohn | Gastric hypomotility in Duchenne's muscular dystrophy[END_REF][START_REF] Bresolin | Cognitive impairment in Duchenne muscular dystrophy[END_REF][START_REF] Nigro | The incidence and evolution of cardiomyopathy in Duchenne muscular dystrophy[END_REF][START_REF] Zellweger | Central nervous system manifestations in childhood muscular dystrophy (CMD). I. Psychometric and electroencephalographic findings[END_REF].

Further features of the disease include a high elevation of serum creatine phosphokinase, myopathic pattern during electromyography, and histological changes including myofiber degeneration, regeneration with fibrosis and fatty infiltrations as well as necrosis accompanied by immune cell infiltrations [START_REF] Emery | Duchenne muscular dystrophy[END_REF][START_REF] Mcdouall | Nature of the mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy[END_REF].

Milder forms of DMD exist and the most common one is the Becker-type muscular dystrophy (BMD, OMIM#300376) [START_REF] Becker | A new x-chromosomal muscular dystrophy[END_REF]. The clinical presentation of BMD patients is much more diverse then in DMD, and walking ability might be lost in the late teens, but in some patients symptoms may occur very late, so that they experience no significant physical impairment. The distribution of muscle wasting and weakness are very similar to DMD [START_REF] Bushby | The clinical, genetic and dystrophin characteristics of Becker muscular dystrophy. I. Natural history[END_REF].

The dystrophin protein, discovered more than 100 years after the first documented cases of DMD as well as its coding dystrophin gene (DMD), which bears the causative mutations, have been subjects to extensive investigations and analysis in the past decades. In DMD patients (Emery and Muntoni, 2003, p14).

mutations disrupt the open reading frame and prevent the expression of dystrophin, whereas in BMD dystrophin is expressed, but in a shorter, internally truncated versions due to in-frame mutations [START_REF] Koenig | The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion[END_REF], however there are exceptions to this reading frame rule.

The long period between the publication of first case reports and the discovery of the disease cause might be due to the fact that the skeletal muscle as the main affected tissue is complex with regard to its development, physiological function and regulation.

The mdx mouse: a naturally occurring animal model for DMD

Animal models are indispensable to study and understand the function of dystrophin in normal and disease states as well as for the development of therapies against DMD. Until present, more than 50 animal models have been described, both laboratory made and naturally occurring ones. Studies have been done using murine, canine and feline models but also small model organisms like the zebrafish Danio rerio (D.rerio) and the nematode Caenorhabditis elegans (C.elegans) [START_REF] Mcgreevy | Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy[END_REF].

The most widely used and best characterized model for DMD is the Dmd mdx mouse, which will be referred to from here on as mdx-mouse, murine dystrophy X-linked [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF].

It has largely contributed to our general knowledge about muscle biology and dystrophin.

The mdx mouse carries a spontaneous nonsense point mutation in exon 23 (CAATAA) at nucleotide position 3185 of Dmd leading to a premature translation termination, (p.Gln1062*) and a subsequent absence of dystrophin expression [START_REF] Sicinski | The molecular basis of muscular dystrophy in the mdx mouse: a point mutation[END_REF]. The mdx mouse displays some hallmarks of DMD-such as muscle degeneration, cardiomyopathy and the elevation of activity of muscle specific enzymes in the serum. However, in contrast to human DMD, those symptoms manifest much later and in milder form if refered to the entire life span of a mouse [START_REF] Quinlan | Evolution of the mdx mouse cardiomyopathy: physiological and morphological findings[END_REF][START_REF] Stedman | The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy[END_REF]. The life span of the mdx mouse is reduced by only 25%, as compared to >50% for DMD patients [START_REF] Mcgreevy | Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy[END_REF]. The dystrophic changes begin to apprear in the animals between 3-6 weeks of life with a rapid muscle fiber degeneration followed by a regeneration phase, typically characterized by the presence of central nuclei in regenerating fibers and an inflammatory reaction. Unlike in humans, where the condition turns rapidly into muscle atrophy, mice maintain hypertroph troughout much of their life span. Only the diaphragm muscle of mdx mice displays the muscle wasting and degeneration observed in DMD boys. [START_REF] Chamberlain | Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma[END_REF][START_REF] Dupont-Versteegden | Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of mdx mice[END_REF][START_REF] Pastoret | mdx mice show progressive weakness and muscle deterioration with age[END_REF][START_REF] Stedman | The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy[END_REF]. The less severe murine phenotype has been partially attributed to an increased expression and probable compensation by utrophin, which is a shorter dystrophin homologue, and will be discussed in more detail in section 4.4.4 (Matsumura et al., 1992;[START_REF] Rybakova | Dystrophin and utrophin bind actin through distinct modes of contact[END_REF].

Consequently, it has been shown that dystrophin/utrophin double mutant mice have a much more severe phenotype than mdx mice [START_REF] Deconinck | Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy[END_REF]. Another explanation for the difference between human and mouse might be a different muscle load and mechanical stress that both organisms are exposed to. If examined under conditions of exhaustion or of physical work, which enhances the in vivo and ex vivo mechanical load, the clinical course of the mdx mouse was shown to be aggravated [START_REF] Danialou | Dystrophin-deficient cardiomyocytes are abnormally vulnerable to mechanical stress-induced contractile failure and injury[END_REF][START_REF] Nakamura | Activation of calcineurin and stress activated protein kinase/p38-mitogen activated protein kinase in hearts of utrophin-dystrophin knockout mice[END_REF].

Furthermore, the body size between man and mouse differ considerably, as do the processes by which such difference is achieved and maintained throughout life [START_REF] Partridge | The mdx mouse model as a surrogate for Duchenne muscular dystrophy[END_REF].

To characterize and understand DMD as a disease process, the function of the dystrophin protein in the muscles and in other tissues has to be understood. In the next sections I thus describe the organization of the muscle and the function which dystrophin has therein. Moreover, I discuss the current knowledge about the role of dystrophin in non-muscle tissue.

The structure and function of the muscle

The muscle is a specialized organ with various functions and locations throughout the body and is characterized by its ability to contract. Based on its histological appearance, we distinguish between smooth and striated muscle, the latter being further subdivided into skeletal and cardiac muscle.

Skeletal muscle

The skeletal muscle is a voluntary controlled muscle and the largest organ of the human body.

In humans, a total of ≈640 skeletal muscles account for ≈38% of total body mass in males and ≈30% in females [START_REF] Janssen | Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr[END_REF]. It is characterized mainly by its mechanical activity required for posture, movement and breathing, which depends on the ability of muscle fibers to contract. The skeletal muscle is composed of parallel bundles of myofibers, which are organized in fascicles while a network of collagen fibers surrounds each fascicle (perimysium) and extends into the space between individual muscle fibers (endomysium). Each myofiber, also called muscle cell, is a multinucleated syncytium generated by fusion of numerous dividing mononucleated myoblasts. After fusion and generation of the fiber, the myonuclei do not divide anymore and remain in the subsarcolemmal space of the sarcoplasm. The muscle stem cells, also called "satellite cells", are mononucleated cells located between the plasma membrane (sarcolemma) and the basement membrane [START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF]. Satellite cells have myogenic potential and the capability to self-renew. They are responsible for postnatal muscle growth, repair and regeneration [START_REF] Emery | Duchenne muscular dystrophy[END_REF].

A single myofiber is composed of many myofibrils containing the contractile proteins actin and myosin, which represent the thin and thick filaments respectively. The myofibrils are organized into sarcomeres, the functional contractile units of the skeletal muscle. The sarcomeres are demarkated by their Z-discs that provide a scaffold for the thin actin filaments and anchor the center of the actin filament bundles. Near the Z-discs thin actin filaments form the I-band. Additional proteins of the Z-discs provide a link to the sarcolemma via the dystrophin-associated protein complex, which plays a central role in the mechanical and contractile properties of the muscle. The region containing the thick myosin filaments is called the A-band and in its center, a dense zone called the M-band is located. Structures called Ttubules are transversely arranged and interconnected tubular extensions of the plasma membrane into the sarcoplasm (Figure 2) [START_REF] Emery | Duchenne muscular dystrophy[END_REF].

Contraction of the skeletal muscle is initiated through an action potential traveling down its supplying nerve (α-motoneuron) leading to a release of the neurotransmitter acetylcholine (ACh) from the presynaptic terminus of the neuromuscular junction (NMJ). In the synaptic cleft of the NMJ, ACh binds to the acetylcholine receptors (AChR) that are densely clustered in the postsynaptic membrane of the NMJ [START_REF] Pratt | Pre-and postsynaptic changes in the neuromuscular junction in dystrophic mice[END_REF]. This results in the depolarization of the skeletal muscle membrane and a downstream release of calcium from the sarcoplasmic reticulum into the cytosol. Calcium causes binding of myosin to actin, which subsequently leads to a contraction of the myofibers as an adenosine triphosphate (ATP) consuming process by which the bundles of myosin filaments slide onto the actin filaments (e.g. the "sliding filament" theory), [START_REF] Huxley | Structural changes in muscle during contraction; interference microscopy of living muscle fibres[END_REF]. This entire process is called "Excitation-contraction coupling".

Myofibers differ in their ability to resist fatigue and produce muscle force. Their fiber type can be determined immunohistochemically via the subtype of its myosin heavy chain. Type I fibers are fatigue resistant and provide little force (also named "slow-twitch", oxidative fibers), type IIa and IIx fibers produce higher force, are less resistant to fatigue and are also called "fasttwitch" and may be either oxidative or glycolytic [START_REF] Schiaffino | Fiber types in mammalian skeletal muscles[END_REF]. Fast IIb fibers are identified in other mammals but not in humans [START_REF] Gorza | Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies[END_REF]. The skeletal muscle is attached to the bone via a tendon and composed of myofibers that are organized into fasciculi. Each myofiber, a multinucleated muscle cell, is surrounded by the plasmalemma and the basal lamina. Sandwiched between these two layers reside the skeletal muscle stem cells, also called the "satellite cells". The myofiber contains numerous myofibrils, actin and myosin filaments which are organized into the sarcomeres. The contractile units consist of actin and myosin filaments. The sarcomeres are divided into alternating I-and Abands giving the fiber their "striated" appearance. Modified from [START_REF] Relaix | Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage[END_REF]

Cardiac and smooth muscle

Cardiac muscle is also striated but an involuntary muscle. Similar as in the skeletal muscle, actin and myosin filaments are organized into sarcomeres, but the contractile tissue has a different structure and is regulated in different manner. The individual cardiomyocytes remain oligonucleated cells, which are connected through the intercalated disc, a highly specialized structure providing mechanical as well as electric coupling. Three different cellcell junctions exist in the intercalated discs: (i) gap junctions, responsible for the electrical coupling, (ii) intermediate filament anchoring desmosomes, and (iii) adherent junctions which anchor actin [START_REF] Perriard | Dilated Cardiomyopathy[END_REF].

Smooth muscles are involuntary as well and their contraction is mostly non-conscious. They are lining the walls of hollow organs like the gastrointestinal tract, uterus, urinary bladder, and blood vessels. The structure of the smooth muscle differs largely from that of skeletal muscle; the muscle cells are thin, elongated and arranged in parallel, which accounts for their smooth appearance. Smooth muscle tissue is primarily under the control of the autonomic nervous system and single cells are connected with each other mechanically through adherens junctions and chemo-electrically through gap junctions.

Myogenesis

The process by which muscles are generated is called "myogenesis". Thereby, we distinguish three phases which are (i) embryonic and fetal development, (ii) postnatal muscle growth, and (iii) re-establishing homeostasis after regeneration. Different factors orchestrate the complex process of myogenesis. All three above mentioned processes are fueled by precursor cells with myogenic potential. Such myocyte-generating cells differ between different muscles, but also between the myogenic phases of the same muscle type.

Skeletal muscles have a remarkable capacity for regeneration after injury, a function attributed to the presence of dormant adult muscle stem cells, the satellite cells, which are also responsible for the postnatal muscle growth. The process of regeneration plays an important role in the pathophysiology of muscle dystrophy, which in itself can be considered a state of chronic injury.

The satellite cell and skeletal muscle regeneration

The satellite cells were first described in 1961 based on their location under the basal lamina of the myofiber [START_REF] Katz | The Terminations of the Afferent Nerve Fibre in the Muscle Spindle of the Frog[END_REF][START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF]. During postnatal growth, the satellite cells proliferate and give rise to myoblasts that differentiate and fuse with each other to form the new myofibers [START_REF] Bischoff | Regeneration of single skeletal muscle fibers in vitro[END_REF][START_REF] Konigsberg | The regenerative response of single mature muscle fibers isolated in vitro[END_REF][START_REF] Lipton | Developmental fate of skeletal muscle satellite cells[END_REF]. When the muscle matures, satellite cells become postmitotic and remain quiescent at their location on the muscle fibers. In the adult muscle, myoblasts are required for normal homeostasis as well as for hypertrophic growth or myofiber repair and regeneration. In those cases, satellite cells are activated, start to proliferate and differentiate thereby supplying the muscle with the necessary myoblasts. As satellite cells have also stem cell properties, they have the ability to self-renew and to replenish their own pool [START_REF] Zammit | The skeletal muscle satellite cell: the stem cell that came in from the cold[END_REF].

The differentiation of the early myoblast through the myogenic program into a mature muscle cell is characterized by a distinct gene expression program. The paired box transcription factor 7 (Pax7) is considered a satellite cell marker, and has been shown to be expressed in mice as well as in many other species including humans [START_REF] Yablonka-Reuveni | The skeletal muscle satellite cell: still young and fascinating at 50[END_REF]. Ablation of Pax7 leads to the inability of the muscle to regenerate thus demonstrating the essential role of satellite cells in this process.

Activation of satellite cells is controlled by proximal signals from the muscle niche and microvasculature and through an inflammatory response [START_REF] Bischoff | Analysis of Muscle Regeneration Using Single Myofibers in Culture[END_REF][START_REF] Carlson | Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells[END_REF][START_REF] Christov | Muscle Satellite Cells and Endothelial Cells: Close Neighbors and Privileged Partners[END_REF][START_REF] Gopinath | Stem Cell Review Series: Aging of the skeletal muscle stem cell niche[END_REF][START_REF] Shavlakadze | Delayed but excellent myogenic stem cell response of regenerating geriatric skeletal muscles in mice[END_REF][START_REF] Yablonka-Reuveni | Fibroblast Growth Factor Promotes Recruitment of Skeletal Muscle Satellite Cells in Young and Old Rats[END_REF]. Furthermore, systemic factors like hepatocyte growth factor, neuronal nitric oxide synthase (nNOS) and Notch-ligands can activate satellite cells [START_REF] Yablonka-Reuveni | The skeletal muscle satellite cell: still young and fascinating at 50[END_REF]. In a states of muscle degeneration such as in DMD, denervation, advanced age, or wasting syndromes, the satellite cell number and their proliferative capacity decreases. The system becomes exhausted thus reducing the regenerative capacity of the muscle [START_REF] Jejurikar | Satellite cell depletion in degenerative skeletal muscle[END_REF]. In DMD, lack of dystrophin and the sarcolemmal instability induce new cycles of degeneration and the satellite cells fail to repair the muscle after unsuccessful rounds of regeneration, which ultimatively results in necrosis. The exact mechanism of reduced regenerative capacity due to satellite cells exhaustion is not clear yet and different theories exist. In mdx mice, it has been shown that the capability to regenerate is not an endogenous quality of satellite cells, but depends on extracellular cues from the stem cell niche [START_REF] Boldrin | Donor Satellite Cell Engraftment Is SignificantlyAugmented When the Host Niche Is Preserved and Endogenous Satellite Cells Are Incapacitated[END_REF][START_REF] Brack | Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis[END_REF][START_REF] Conboy | Rejuvenation of aged progenitor cells by exposure to a young systemic environment[END_REF]. Boldrin and colleagues demonstrated that satellite cells from young and aged mdx mice can regenerate in vivo in the same manner as wildtype satellite cells (Boldrin et al., 2015). Other studies show that cellautonomous factors (satellite cell intrinsic functions) are responsible for the changes in their regenerative capacity [START_REF] Cosgrove | Rejuvenation of the muscle stem cell population restores strength to injured aged muscles[END_REF][START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF]. In a recent study Dumont and colleagues suggested that the impaired regeneration in mdx mice was the result of defective asymmetric division of satellite cells and generation of myogenic progenitors due to intrinsic dysfunction resulting from dystrophin loss [START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF]. The aforementioned work described for the first time a functional role of dystrophin in satellite cells and is discussed in section 4.4.7. Another recent study showed in mice that, unlike previously assumed, loss of satellite cell-dependant regenerative capacity had no direct impact on sarcopenia, the state of age-related loss of muscle mass and strength. However, the satellite cell loss contributed to the increased fibrosis observed in aged skeletal muscle [START_REF] Fry | Inducible depletion of satellite cells in adult, sedentary mice impairs muscle regenerative capacity without affecting sarcopenia[END_REF]. These studies highlight the necessity of a broader research approach, which should be aimed to unravel the interplay of cell intrinsic and extrinsic factors that influence the satellite cell fate in normal state and conditions of degeneration. Such understaning could be exploited for the development of satellite cell centered regenerative therapies.

Dystrophin

The DMD gene

Dystrophin is a large, rod-shaped protein which is expressed in all types of muscle (i.e. skeletal, cardiac, and smooth muscles) as well as in neurons [START_REF] Hoffman | Dystrophin: The protein product of the duchenne muscular dystrophy locus[END_REF][START_REF] Hoffman | Cell and fiber-type distribution of dystrophin[END_REF][START_REF] Uchino | Localisation and characterisation of dystrophin in the central nervous system of controls and patients with Duchenne muscular dystrophy[END_REF]. As already mentioned it is encoded by the DMD gene, which causes Duchenne muscular dystrophy if mutated [START_REF] Koenig | Complete cloning of the duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF]A. P. Monaco et al., 1986).

The dystrophin gene (DMD) is the largest human gene spanning 2.4 Mb at the Xp21 locus and comprises 79 primary exons plus 6 alternative first exons [START_REF] Emery | Duchenne muscular dystrophy[END_REF].

However, the coding sequence is only ≈14 kb, less than 1% of its genomic DNA sequence [START_REF] Hoffman | Dystrophin: The protein product of the duchenne muscular dystrophy locus[END_REF][START_REF] Koenig | Complete cloning of the duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF]Anthony P. Monaco et al., 1986) mRNAs for several dystrophin isoforms are transcribed from the DMD gene through the use of tissue specific promoters or by alternative splicing. The full-length dystrophin protein has a molecular weight of 427 kDa and the corresponding mRNA is transcribed from 3 different tissue specific promoters: muscle (M), brain (B) and Purkinje cell type (P). The main dystrophin isoform Dp427 is present in the skeletal, cardiac and smooth muscle and is controlled by the muscle specific promoter. It carries a unique N-terminal sequence, which is encoded by exon 1 [START_REF] Bies | Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development[END_REF]. This promoter is also active in glial cells [START_REF] Chelly | Dystrophin gene transcribed from different promoters in neuronal and glial cells[END_REF]. The brain promoter (B) is active in cortical neurons, heart-and cerebellar neurons, while the Purkinje cell specific promoter regulates the cerebellar dystrophin expression [START_REF] Górecki | Expression of four alternative dystrophin transcripts in brain regions regulated by different promoters[END_REF][START_REF] Nudel | Duchenne muscular dystrophy gene product is not identical in muscle and brain[END_REF].

Developmentally regulated shorter dystrophin isoforms are transcribed from internal promoters like the retinal dystrophin Dp260 spanning exons 30-79 , the cerebellar and renal isoforms Dp140 spanning exons 45-79 [START_REF] Lidov | Dp140: a novel 140 kDa CNS transcript from the dystrophin locus[END_REF], the Schwann cell Dp116 dystrophin spanning exons 56-79. These three isoforms (Dp260, Dp116 and Dp140) comprise parts of the rod domain and express the cysteine-rich and C-terminal domains, but lack the N-terminal actin binding domain [START_REF] Byers | An alternative dystrophin transcript specific to peripheral nerve[END_REF][START_REF] Souza | A novel dystrophin isoform is required for normal retinal electrophysiology[END_REF][START_REF] Lidov | Dp140: a novel 140 kDa CNS transcript from the dystrophin locus[END_REF].

A more ubiquitously expressed short isoform is Dp71, which spans exons 63-79 (Figure 3). It has a unique seven residue N-terminus fused to the cysteine-rich and C-terminal domains [START_REF] Bar | A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution[END_REF][START_REF] Howard | Cloning and characterization of alternatively spliced isoforms of Dp71[END_REF][START_REF] Lederfein | A 71-kilodalton protein is a major product of the Duchenne muscular dystrophy gene in brain and other nonmuscle tissues[END_REF][START_REF] Lederfein | A housekeeping type promoter, located in the 3'region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene[END_REF].

Further dystrophin diversification is provided by alternative splicing throughout the coding sequence of dystrophin [START_REF] Bies | Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development[END_REF][START_REF] Sironi | The dystrophin gene is alternatively spliced throughout its coding sequence[END_REF]. Notably, two alternative splicing sites exist at the 3' end of dystrophin [START_REF] Feener | Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus[END_REF]. Their usage has been well characterized in the Dp71 isoform. In Dp71d (also named Dp71a) splicing of exon 71 does not change the reading frame of the transcript and generates the 13 amino acid long C-terminus common to most dystrophin isoforms. The Dp71f (Dp71b) isoform is generated by alternative splicing of exon 78 which causes a change in the reading frame and produces a C-terminus that contains 31 new amino acids with hydrophobic properties. Another isoform is Dp71c lacking exon 71-74 that encode the 110 amino acid sequence of the syntrophin binding domain. Moreover, exon 78 can be further skipped in the latter isoform creating the Dp71Δ110 variant. The expression of these isoforms is differentially regulated during human embryonic development and adulthood [START_REF] Austin | Cloning and characterization of alternatively spliced isoforms of Dp71[END_REF][START_REF] Bies | Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development[END_REF][START_REF] Feener | Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus[END_REF]. Similar expression patterns have been observed in animal models on the mRNA and protein level [START_REF] Bies | Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development[END_REF][START_REF] Daoud | Analysis of Dp71 contribution in the severity of mental retardation through comparison of Duchenne and Becker patients differing by mutation consequences on Dp71 expression[END_REF].

The various isoforms and splicing variants of dystrophin are highly conserved in mammals and differ in their cellular and subcellular localization as well as in binding partners thus contributing to the pleiotropic functions of dystrophin. is present in all isoforms, contains binding sites for proteins of the dystrophin associated protein complex (DAPC). The cystein-rich domain includes the WW binding domain, which mediates specific protein-protein interactions with short proline-rich or proline-containing motif, the ZZ-type-zinc finger binding domain and a coiled-coil domain in the C-terminus. Modified from [START_REF] Blake | The neurobiology of duchenne muscular dystrophy: learning lessons from muscle?[END_REF].

The dystrophin protein in the skeletal muscle

Only full-length dystrophin is expressed in neonatal and adult skeletal muscle where it accounts for 0,002% of the total protein content. In striated muscles, dystrophin is localized at the cytoplasmatic side of the sarcolemma [START_REF] Watkins | Immunoelectron microscopic localization of dystrophin in myofibres[END_REF]. In longitudinal sections, dystrophin expression shows a striated pattern and co-localizes with the costamers, when the skeletal muscle fibers are visualized [START_REF] Ervasti | Costameres: the Achilles' heel of Herculean muscle[END_REF][START_REF] Porter | Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle[END_REF][START_REF] Straub | Direct visualization of the dystrophin network on skeletal muscle fiber membrane[END_REF]. Furthermore, dystrophin is concentrated around the NMJ [START_REF] Huard | Is dystrophin present in the nerve terminal nat the neuromuscular junction? An immunoshistochemical study of the heterozygote dystrophic (mdx) mouse[END_REF]. The full-length dystrophin isoform has a molecular weight (MW) of 427 kDa and is composed of 3,685 amino acids. At the sarcolemma, dystrophin assembles several transmembrane and cytoplasmic proteins into the dystrophin associated protein complex (DAPC). Via this complex, dystrophin A B provides a link between the cytoskeleton and the extracellular matrix (ECM) that assures mechanical stability of the sarcolemma and protects the muscle from contraction induced muscle damage.

Dystrophin belongs to the β-spectrin/ α-actinin protein family [START_REF] Koenig | The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein[END_REF] and can be divided into 4 functional domains: an N-terminal actin binding domain, a central roddomain, a cysteine-rich, and a C-terminal domain (Figure 3). The N-terminal and part of the dystrophin rod domain interact with cytoskeletal actin [START_REF] Amann | A cluster of basic repeats in the dystrophin rod domain binds F-actin through an electrostatic interaction[END_REF][START_REF] Norwood | The structure of the N-terminal actin-binding domain of human dystrophin and how mutations in this domain may cause Duchenne or Becker muscular dystrophy[END_REF]. The rod domain folds into α-helical coils composed of 24 spectrin-like repeats interrupted by 4 proline-rich hinges that contribute to the flexible structure of the protein [START_REF] Blake | Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle[END_REF][START_REF] Koenig | The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein[END_REF]. Behind the hinge region we find a WW protein binding domain, which can also be found in signaling and regulatory molecules [START_REF] Bork | The WW domain: a signalling site in dystrophin?[END_REF].

This WW-domain binds to β-dystroglycan, which in turn connects the DAPC to α-laminin in the ECM, an interaction mediated by α-dystroglycan [START_REF] Judge | Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex[END_REF]. The WW-domain is followed by the cysteine-rich domain displaying two EF hand motifs that are potential binding sites for calcium ions [START_REF] Koenig | The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein[END_REF]. The ability of dystrophin to bind calcium in a calmodulin dependent manner is facilitated by the ZZ-motif [START_REF] Anderson | Ca2+-calmodulin binds to the carboxyl-terminal domain of dystrophin[END_REF], which is enriched with cysteine residues. These are predicted to form the coordination sites for binding divalent metal cations such as zinc ions [START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF]. In the C-terminal domain, another coil-coil region of dystrophin interacts with syntrophin that can bind to nNOS and to dystrobrevin.

A major role of the dystrophin protein is the assembly of the DAPC. Hence, dystrophin provides a link between the cytoskeleton and the extracellular matrix. Absence of this mechanical apparatus is thought to destabilize the sarcolemma, making the myofibers more susceptible to damage during contractions [START_REF] Ehmsen | The dystrophin-associated protein complex[END_REF][START_REF] Lederfein | A housekeeping type promoter, located in the 3'region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene[END_REF][START_REF] Pasternak | Mechanical function of dystrophin in muscle cells[END_REF][START_REF] Petrof | Dystrophin protects the sarcolemma from stresses developed during muscle contraction[END_REF][START_REF] Sacco | Contractile properties and susceptibility to exercise-induced damage of normal and mdx mouse tibialis anterior muscle[END_REF]. Another important role of dystrophin is its scaffolding function for signal transduction through the DAPC and for various ion channels. DAPC members either bind to or are substrates for known signaling molecules like nNOS, caveolin3, growth factor receptor-bound protein 2 (Grb2) and various kinases [START_REF] Rando | The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies[END_REF][START_REF] Sotgia | Caveolin-3 directly interacts with the C-terminal tail of beta -dystroglycan. Identification of a central WW-like domain within caveolin family members[END_REF][START_REF] Sotgia | Tyrosine phosphorylation of beta-dystroglycan at its WW domain binding motif, PPxY, recruits SH2 domain containing proteins[END_REF][START_REF] Spence | Dystroglycan, a scaffold for the ERK-MAP kinase cascade[END_REF][START_REF] Yang | SH3 Domain-mediated Interaction of Dystroglycan and Grb2[END_REF]. Dystrophin plays a role in the cytosolic calcium homeostasis, which is impaired in dystrophic muscle due to an alteration of ion channels and pumps [START_REF] Friedrich | L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle[END_REF]. Dystrophin has also a function at the NMJ. Hence, in dystrophic muscle the post-synaptic membrane shows reduced folding, which disturbs the distribution of AChRs and of other proteins at the post-synaptic membrane. This observation is most likely due to defects in nNOS localization or nitric oxide (NO) production [START_REF] Shiao | Defects in neuromuscular junction structure in dystrophic muscle are corrected by expression of a NOS transgene in dystrophin-deficient muscles, but not in muscles lacking α-and β1-syntrophins[END_REF].

Most of the studies about functional domains of dystrophin and its binding partners have been conducted using transgenic mice that over-expressed mini-or micro-dystrophins, which contained various functional domains (Cox et al., 1994a;[START_REF] Gaedigk | Improvement in survival and muscle function in an mdx/utrn(-/-) double mutant mouse using a human retinal dystrophin transgene[END_REF][START_REF] Harper | Modular flexibility of dystrophin: Implications for gene therapy of Duchenne muscular dystrophy[END_REF][START_REF] Judge | Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex[END_REF][START_REF] Lai | Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy[END_REF][START_REF] Tinsley | Expression of full-length utrophin prevents muscular dystrophy in mdx mice[END_REF][START_REF] Warner | Expression of Dp260 in muscle tethers the actin cytoskeleton to the dystrophin-glycoprotein complex and partially prevents dystrophy[END_REF].

The mechanical and signaling functions of dystrophin are exerted through the interaction with a complex protein network at the sarcolemma, the DAPC, which will be described in more detail in the next section.

Dystrophin associated protein complex (DAPC)

Dystrophin serves as the central player for assembling the DAPC at the sarcolemma. The complex provides the mechanical link from actin to the ECM and recruits either directly or indirectly signaling molecules. The importance of dystrophin in assembling the complex is highlighted by the absence of DAPC in dystrophic muscle. The DAPC components can be divided into the following subcomplexes: the dystroglycan complex, the sarcoglycan complex, as well as the cytoplasmic and ECM components including sarcospan, syntrophin, dystrobrevin and nNOS (Figure 4) [START_REF] Blake | The neurobiology of duchenne muscular dystrophy: learning lessons from muscle?[END_REF][START_REF] Ehmsen | The dystrophin-associated protein complex[END_REF]. Beyond that, several components of the DAPC are also found in the CNS generating various DAPC-like complexes.

The dystroglycan complex (α-and β-dystroglycan) provides the core of the DAPC. The αdystroglycan is located in the extracellular matrix where it interacts with laminins 1, 2 and 4, perlecan and agrin and is required for the assembly of the basal laminae. At the NMJ, dystroglycans plays a specialized role where it binds to agrin and laminin 4, two proteins required for NMJ formation. Furthermore α-dystroglycan serves as a cell surface receptor for a number of micro-organisms [START_REF] Hemler | Dystroglycan Versatility[END_REF][START_REF] Ibraghimov-Beskrovnaya | Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix[END_REF]. βdystroglycan spans the sarcolemma and binds with its proline-rich C-terminus to the WWand EF-domains of dystrophin as well as to the SH3-domain of Grb2. Such and interaction could transmit extracellular signals to the muscle cytoskeleton [START_REF] Yang | SH3 Domain-mediated Interaction of Dystroglycan and Grb2[END_REF]. Both dystroglycans are produced from a single polypeptide and are heavily glycosylated prior to being sorted to their respective location [START_REF] Winder | The complexities of dystroglycan[END_REF]. In mice, dystroglycan function is indispensable for survival. Total absence is embryonically lethal due to disruption of extraembryonic basement membrane formation [START_REF] Williamson | Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag1-null mice[END_REF]. In the CNS, conditional deletion of dystroglycans leads to impaired synaptic plasticity [START_REF] Moore | Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy[END_REF]. Moreover, genetic loss or defective glycosylation of α-dystroglycan leads to alterations of the basement membrane and abnormal neural migration during development [START_REF] Perronnet | Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies, Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies[END_REF].

These findings point out the crucial functions of dystroglycans in non-muscle tissues.

The sarcoglycan complex consists of 6 transmembrane proteins (α-, β-, γ-, δ-, ε-ζsarcoglycans) and only the γ-sarcoglycan directly interacts with dystrophin. δ-Sarcoglycan is located closer to the dystroglycan complex and forms a tight link with β-sarcoglycan [START_REF] Chan | Molecular organization of sarcoglycan complex in mouse myotubes in culture[END_REF]. Mutations in each of the sarcoglycans lead to a specific form of muscular dystrophy, the so called Limb Girdle Muscular Dystrophy. This disease entity is associated with a reduction or absence of the other DAPC members at the sarcolemma. Mutant sarcoglycans are thought to block the formation of the complex and its insertion into the sarcolemma thus leading to a dystrophic phenotype [START_REF] Holt | Assembly of the sarcoglycan complex. Insights for muscular dystrophy[END_REF]. Further functional roles of the sarcoglycans have been derived from its structure: γ-sarcoglycan has 5 tyrosine residues in its cytoplasmic domain suggesting bidirectional signaling with integrins [START_REF] Yoshida | Bidirectional signaling between sarcoglycans and the integrin adhesion system in cultured L6 myocytes[END_REF].

Until recently, the expression of sarcoglycans was thought to be restricted to the striated and smooth muscles and to lesser extent to the peripheral nerves. However, recent research has shown that ε-sarcoglycan plays an important role in the brain and that ζ-sarcoglycan is abundantly expressed in the brain as well [START_REF] Shiga | Zeta-sarcoglycan is a functional homologue of gamma-sarcoglycan in the formation of the sarcoglycan complex[END_REF][START_REF] Zimprich | Mutations in the gene encoding epsilonsarcoglycan cause myoclonus-dystonia syndrome[END_REF].

Sarcospan is a unique transmembrane protein whose N-and C-termini are both located intracellularly. It forms a sub-complex with the sarcoglycans [START_REF] Crosbie | Sarcospan, the 25-kDa transmembrane component of the dystrophin-glycoprotein complex[END_REF][START_REF] Ehmsen | The dystrophin-associated protein complex[END_REF].

The C-terminal coiled coil domain of dystrophin represents the binding site for the cytoplasmic components of the DAPC. The syntrophin family is composed of five subtypes (α, β1, β2, γ1, and γ2 syntrophin) that are expressed and differently distributed in the muscle. αsyntrophins are localized at the sarcolemma of all fibers, β1-syntrophins are present in fasttwitch fibers, and β2-syntrophins can be found at the NMJ. Syntrophins are characterized by two plekstrin homology (PH) domains and a PDZ domain (PSD-95, postsynaptic densitiy protein 95; disc-large and ZO-1, zonula occludens-1) domain. They bind directly to dystrophin and to α-dystrobrevin, both of which having two syntrophin binding sites at their C-terminus [START_REF] Newey | Alternative splicing of dystrobrevin regulates the stoichiometry of syntrophin binding to the dystrophin protein complex[END_REF]. The ErbB4 receptor tyrosine kinase, voltage gated sodium channels, nNOS, stress-activated protein kinases (SAPK), aquaporin-4 (AQP4) and neuroligins, have been proposed as potential binding partners for syntrophins via their PDZ domain [START_REF] Brenman | Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy[END_REF][START_REF] Brenman | Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains[END_REF][START_REF] Garcia | The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses[END_REF][START_REF] Gee | Interaction of Muscle and Brain Sodium Channels with Multiple Members of the Syntrophin Family of Dystrophin-Associated Proteins[END_REF][START_REF] Hasegawa | Stress-activated Protein Kinase-3 Interacts with the PDZ Domain of α1-Syntrophin A MECHANISM FOR SPECIFIC SUBSTRATE RECOGNITION[END_REF][START_REF] Schultz | Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels[END_REF][START_REF] Yamakawa | Neuroligins 3 and 4X interact with syntrophin-gamma2, and the interactions are affected by autism-related mutations[END_REF]. Many of the aforementioned syntrophin interaction partners have important functions in the brain, however, the effects they have on the DAPC-like complexes remains to be elucidated.

Dystrobrevin has five different isoforms, which are generated through alternative splicing and through different tissue specific promoters in a similar manner as already described for dystrophin. Two of the isoforms are highly expressed at the sarcolemma where they associate with syntrophin. In skeletal muscle, α-dystrobrevin-2 is the primary isoform associated with the DAPC, and α-dystrobrevin-1 is mainly localized at the NMJ, where it interacts with utrophin [START_REF] Peters | Differential Membrane Localization and Intermolecular Associations of α-Dystrobrevin Isoforms in Skeletal Muscle[END_REF]. Moreover, dystrobrevins interacts with syncoilin, which is highly expressed at the sarcolemma of skeletal, cardiac and smooth muscles. In addition, syncoilin co-localizes with desmin at the NMJ, Z-lines and sarcolemma of skeletal muscle hence providing connection between the DAPC with the intermediate filaments system [START_REF] Poon | Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex[END_REF]. β-dystrobrevin is present in brain, but not in muscle, and can bind directly to dystrophin. It is exclusively expressed in neurons and there expressed at high levels at the postsynaptic densities (PSD) [START_REF] Blake | beta-dystrobrevin, a member of the dystrophin-related protein family[END_REF][START_REF] Blake | Different Dystrophin-like Complexes Are Expressed in Neurons and Glia[END_REF][START_REF] Peters | beta-dystrobrevin, a new member of the dystrophin family. Identification, cloning, and protein associations[END_REF]. Dystrophin interacts with actin through its N-terminal domain and part of the rod domain. 24 spectrin repeats compose the rod domain and are followed by a WW protein binding domain, which interacts with βdystroglycan. The latter connects to laminin at the ECM via α-dystroglycan. The dystroglycan complex at the sarcolemma interacts with the other big transmembrane complex, which is composed of sarcoglycans. Sarcospan covers the sarcoglycan complex towards the ECM. At the C-terminus dystrophin reacts via its cysteinerich domain with syntrophins that in turn can bind to α-dystrobrevin, nNOS and caveolin-3 [START_REF] Davies | Molecular mechanisms of muscular dystrophies: old and new players[END_REF].

Utrophin: a dystrophin homolog

Utrophin is a structural homolog of dystrophin and contains the same protein binding domains, although small differences exist; utrophin lacks the actin binding part, which is located in the rod domain of dystrophin. Utrophin is the autosomally encoded homolog of dystrophin. During embryonic development it is expressed at the sarcolemma of the fetal muscle and becomes gradually replaced by dystrophin postnatally [START_REF] Clerk | Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle[END_REF]. It is assumed that dystrophin is better specialized and adapted for the higher workload of skeletal muscle and heart during birth and thereafter [START_REF] Tinsley | Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene[END_REF]. In the adult muscle utrophin is expressed in intramuscular nerves, blood vessels, and in the muscle fibers, where it is localized at the NMJ and at the myotendinous junction [START_REF] Ohlendieck | Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle[END_REF]. Utrophin is thought to be part of the postsynaptic cytoskeleton [START_REF] Love | Dystrophin and dystrophin-related proteins: a review of protein and RNA studies[END_REF]. Both dystrophin and utrophin, however, are found at different locations of the NMJ, blood vessels, and cardiac muscle [START_REF] Pons | Utrophin localization in normal and dystrophin-deficient heart[END_REF]. In myopathies, the expression of utrophin is increased at the sarcolemma and it is upregulated in regenerating fibers [START_REF] Helliwell | The dystrophin-related protein, utrophin, is expressed on the sarcolemma of regenerating human skeletal muscle fibres in dystrophies and inflammatory myopathies[END_REF]. The upregulation of utrophin in mdx mice could partly compensate for the absence of dystrophin and ameliorate muscle pathology. In muscle dystrophic mice, utrophin overexpression via transgene or cDNA delivery and gutted adenovirus improved dystrophic pathology in the animals. Utrophin has been shown to provide sarcolemmal stability and restore the DAPC to some extent, but the nNOS expression was not restored suggesting that full-length utrophin cannot anchor nNOS at the sarcolemma [START_REF] Gilbert | Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles[END_REF][START_REF] Li | Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin[END_REF][START_REF] Tinsley | Expression of full-length utrophin prevents muscular dystrophy in mdx mice[END_REF]. Therapies aiming at the upregulation of the endogenous utrophin in DMD patients have been subject of many investigations. A small molecule (BMN195) was identified that could modulate the expression of utrophin. In pre-clinical models it showed promise to upregulate utrophin [START_REF] Tinsley | Daily Treatment with SMTC1100, a Novel Small Molecule Utrophin Upregulator, Dramatically Reduces the Dystrophic Symptoms in the mdx Mouse[END_REF]. Nevertheless, a clinical study failed to confirm a clinical benefit due to poor bioavailability (http://www.musculardystrophyuk.org/pressreleases/news2568disappointing_results_from_biomarin_s_utrophin_clinical_trial_for_duc henne/). Presently additional candidates that should overcome the limitations of BMN195, but with a similar positive effect on utrophin upregulation are being developed [START_REF] Tinsley | Future Clinical And Biomarker Development For SMT C1100, The First Utrophin Modulator To Enter Clinical Trials For Duchenne Muscular Dystrophy (DMD) (S6.004)[END_REF].

Dystrophin in cardiac and smooth muscles

The important role of dystrophin in cardiac and smooth muscles is highlighted by the fact that most of DMD patients die from cardiac and respiratory failure. Patients do not only suffer from skeletal muscle related symptoms but also from cardiac [START_REF] Emery | Abnormalities of the electrocardiogram in hereditary myopathies[END_REF][START_REF] Perloff | The distinctive electrocardiogram of Duchenne's progressive muscular dystrophy. An electrocardiographic-pathologic correlative study[END_REF] and vascular dysfunction [START_REF] Noordeen | Blood loss in Duchenne muscular dystrophy: vascular smooth muscle dysfunction?[END_REF] as well as dysfunction of the gastrointestinal tract [START_REF] Barohn | Gastric hypomotility in Duchenne's muscular dystrophy[END_REF][START_REF] Chung | Acute gastroparesis in Duchenne's muscular dystrophy[END_REF][START_REF] Leon | Chronic intestinal pseudoobstruction as a complication of Duchenne's muscular dystrophy[END_REF].

The muscle (DP427m) and brain specific (Dp427b) dystrophin isoforms are the major ones in heart and smooth muscle. During development, the dystrophin expression at the plasmalemma occurs earlier in cardiac and smooth foetal muscle (8 weeks of gestation) then in skeletal muscle. Of note, in cardiac muscle the brain and muscle isoforms are expressed in parallel [START_REF] Torelli | Expression, regulation and localisation of dystrophin isoforms in human foetal skeletal and cardiac muscle[END_REF].

Dystrophin in the heart muscle

In cardiac myocytes, dystrophin is localized at the sarcolemma and the T-tubules [START_REF] Klietsch | Dystrophin-glycoprotein complex and laminin colocalize to the sarcolemma and transverse tubules of cardiac muscle[END_REF]. In contrast to skeletal muscle, where loss of dystrophin dramatically reduces the expression of other DAPC components, their expression in the heart remains unaffected [START_REF] Townsend | Systemic administration of micro-dystrophin restores cardiac geometry and prevents dobutamine-induced cardiac pump failure[END_REF]. The reason for this striking difference is not yet known. In DMD patients, the primary cellular defect, leading to the demise of cardiac myocytes and subsequent cardiomyopathy, is thought to be the mechanical instability of the cell membrane [START_REF] Fanchaouy | Pathways of abnormal stress-induced Ca2+ influx into dystrophic mdx cardiomyocytes[END_REF][START_REF] Yasuda | Dystrophic heart failure blocked by membrane sealant poloxamer[END_REF]. These findings confirm the observation from skeletal muscle that the primary role of dystrophin is the mechanical stabilization of the sarcolemma. It has been shown that isolated cardiac myocytes from mdx mice are more susceptible to membrane damage due to either passive stretch or hypo-osmotic stress. Like in skeletal muscles, this mechanical injury is associated with increased membrane permeability, decreased force production, lateral force transmission, but not with reductions in the force generating capacity of the sarcomere [START_REF] Fanchaouy | Pathways of abnormal stress-induced Ca2+ influx into dystrophic mdx cardiomyocytes[END_REF][START_REF] Yasuda | Dystrophic heart failure blocked by membrane sealant poloxamer[END_REF].

In addition, abnormalities of dystrophin expression have not only been observed in inherited dystrophinopathies but also in non-inherited conditions such as acute ischemia, enteroviral infection, and isoproterenol-induced cardiomyopathy [START_REF] Armstrong | Ischemic Loss of Sarcolemmal Dystrophin and Spectrin: Correlation with Myocardial Injury[END_REF][START_REF] Kyoi | Dystrophin is a possible end-target of ischemic preconditioning against cardiomyocyte oncosis during the early phase of reperfusion[END_REF][START_REF] Toyo-Oka | Evidence that Activation of Endogenous Calpain is Responsible for the Pathogenesis of Ischemic Cardiomyopathy via Loss of Dystrophin and its Associated Proteins[END_REF]. It has been shown in mice that dystrophin expression in the heart declines significantly with age [START_REF] Bodyak | Gene expression profiling of the aging mouse cardiac myocytes[END_REF]. Furthermore, under conditions of isoprotenerol intoxication and ischemia, dystrophin has an enhanced susceptibility to degradation in contrast to integrins and other DAPC proteins [START_REF] Campos | Isoproterenol induces primary loss of dystrophin in rat hearts: correlation with myocardial injury[END_REF][START_REF] Rodríguez | Ischemia depletes dystrophin and inhibits protein synthesis in the canine heart: mechanisms of myocardial ischemic injury[END_REF]. Degradation of dystrophin is achieved through calpain proteases, which are calcium-dependent cysteine proteinases. These enzymes can be aberrantly activated in DMD and heart failure [START_REF] Takahashi | Possible involvement of calpain activation in pathogenesis of chronic heart failure after acute myocardial infarction[END_REF][START_REF] Zatz | Calpains and disease[END_REF]. In transgenic mdx mice, overexpression of the calpain inhibitor calpistatin led to reduction in muscle necrosis. Hence, expression of the endogenous calpain inhibitor calpistatin and of other inhibitors might be a therapeutic strategy for DMD and during heart failure [START_REF] Spencer | Overexpression of a calpastatin transgene in mdx muscle reduces dystrophic pathology[END_REF].

Dystrophin can also be cleaved by the 2A proteases that are expressed by cardiotropic enteroviruses [START_REF] Badorff | Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy[END_REF]. Cleavage disconnects the actin binding and rod domain from the C-terminus, thereby contributing to the destabilization of the DAPC and the sarcolemma.

Dystrophin in the smooth muscle

DMD patients often suffer from symptoms such as dysphagia, vomiting, gastric dilation, paralytic ileus, severe constipation and bladder paralysis in the later phases of their disease.

Most of the information about smooth muscle pathology has been gained by autopsies, where small fiber sizes, atrophy or loss of myofibers, and fibrosis have been noted in gastrointestinal smooth muscles [START_REF] Barohn | Gastric hypomotility in Duchenne's muscular dystrophy[END_REF][START_REF] Chung | Acute gastroparesis in Duchenne's muscular dystrophy[END_REF][START_REF] Jaffe | Symptoms of upper gastrointestinal dysfunction in Duchenne muscular dystrophy: case-control study[END_REF][START_REF] Leon | Chronic intestinal pseudoobstruction as a complication of Duchenne's muscular dystrophy[END_REF][START_REF] Nowak | Gastrointestinal manifestations of the muscular dystrophies[END_REF].

Dystrophin in smooth muscles is found at distinct regions of their plasmamembrane.

Dystrophin localizes at the caveolae-rich membrane domains that lie between the ribs of the adherens junctions, together with caveolin, the inositol triphosphate (IP3) receptor and the sodium-calcium-ion exchanger [START_REF] North | Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle[END_REF].

A study on vascular smooth muscles shows that dystrophin plays an important role in smooth muscle function and differentiation. The dystrophin gene was among the genes whose transcription was induced by actin polymerization and its expression was promoted by the transcription factors myocardin and myocardin-related transcription factor A [START_REF] Turczyńska | Regulation of smooth muscle dystrophin and synaptopodin 2 expression by actin polymerization and vascular injury[END_REF]. Contractile smooth muscle cells of the vascular system show high degree of plasticity and can undergo reversible phenotypic changes in response to environmental cues and vascular injury. An important function in these processes is attributed to the DAPC by linking the actin filaments to the ECM. A strong correlation between dystrophin expression and the contractile performance of the vascular smooth muscle has been shown in vivo and in vitro. However, in the presence of serum, dystrophin is not expressed in cultured smooth muscle cells, which causes their dedifferentiation [START_REF] Lees | Dystrophin (Xp21), a New Phenotype Marker of Cultured Rat Aortic Myocytes[END_REF][START_REF] Lees | Parallel expression level of dystrophin and contractile performances of rat aortic smooth muscle[END_REF].

Dystrophin in non-muscle tissues

The role of dystrophin in muscle has been intensively investigated in DMD patients. The absence of dystrophin from the sarcolemma triggers a cascade of events leading to muscle degeneration [START_REF] Emery | Duchenne muscular dystrophy[END_REF]. However, DMD can be considered a multi-systemic disorder and dysfunction of the numerous dystrophin isoforms has pleiotropic effects in nonmuscle tissues. DMD patients do not only suffer from muscle related symptoms, but display CNS dysfunction including cognitive impairment [START_REF] Dubowitz | The central nervous system in Duchenne muscular dystrophy[END_REF][START_REF] Mehler | Brain dystrophin, neurogenetics and mental retardation[END_REF][START_REF] Yoshioka | Central nervous system involvement in progressive muscular dystrophy[END_REF] as well as impairment of the visual system [START_REF] Cibis | The effects of dystrophin gene mutations on the ERG in mice and humans[END_REF][START_REF] Costa | Red-Green Color Vision Impairment in Duchenne Muscular Dystrophy[END_REF][START_REF] Souza | A novel dystrophin isoform is required for normal retinal electrophysiology[END_REF][START_REF] Pillers | Dystrophin expression in the human retina is required for normal function as defined by electroretinography[END_REF].

In the nervous system all dystrophin isoform have been identified and are expressed in adult life but also throughout neural development. During development, dystrophin is expressed within the neural tube, selected areas of the embryonic and postnatal neuraxis suggesting a possible regulatory role in neurogenesis, neuronal migration and cellular differentiation [START_REF] Ceccarini | A splice variant of Dp71 lacking the syntrophin binding site is expressed in early stages of human neural development[END_REF][START_REF] Cisneros | Expression of dystrophin Dp71 during PC12 cell differentiation[END_REF][START_REF] Sarig | Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development[END_REF][START_REF] Schofield | Apo-dystrophin-1 and apo-dystrophin-2, products of the Duchenne muscular dystrophy locus: expression during mouse embryogenesis and in cultured cell lines[END_REF]. During this period, the most prominent isoform is the shortest Dp71. In adulthood, the brain expresses the highest variety of isoforms, whereas Dp260 is restricted to the retina (D 'Souza et al., 1995) and the Dp116 isoform is found predominantly in Schwann cells of peripheral nerves [START_REF] Byers | An alternative dystrophin transcript specific to peripheral nerve[END_REF].

The exact role of dystrophin in the CNS as well as its correlation to the CNS phenotype in DMD still remains to be elucidated and is hampered by the complexity and high variety of dystrophin proteins and DAPC-like complexes expressed throughout development and adult life.

Dystrophin in the brain

Although dystrophin levels in the brain are only 10% of those in muscles, the highest number of isoforms can be found there. Observations in some DMD patients with impairment of cognition or memory, as well as behavioral problems have focused the research towards the role of dystrophin in the brain. Interestingly, a correlation can be found between the position of a mutation in the DMD gene and the presence of cognitive problems. All DMD patients display moderate but specific memory and attention deficits. Since Dp427 isoforms is commonly lost in all DMD patients, a role of full-length dystrophin in brain dysfunction has been suggested. However, the severity and frequency of mental retardation increases with a progressive loss of functional C-terminal isoforms [START_REF] Bushby | Genetic and clinical correlations of Xp21 muscular dystrophy[END_REF][START_REF] Lenk | Point mutations at the carboxy terminus of the human dystrophin gene: implications for an association with mental retardation in DMD patients[END_REF]. Mutations extending to exon 50 leading to loss of Dp427, Dp260 and Dp140 have been shown to contribute significantly to mental retardation in DMD patients [START_REF] Bardoni | Loss of Dp140 regulatory sequences is associated with cognitive impairment in dystrophinopathies[END_REF][START_REF] Emery | Duchenne muscular dystrophy[END_REF][START_REF] Rapaport | Apparent association of mental retardation and specific patterns of deletions screened with probes cf56a and cf23a in Duchenne muscular dystrophy[END_REF]. The major features of CNS involvement in DMD have been observed and reported already in the 19 th century by Duchenne describing a seven year old boy as dull and with poor language skills [START_REF] Duchenne | Recherches sur la paralysie musculaire pseudohypertrophique ou paralysie myo-sclérosique[END_REF]. The mental retardation found in 20-30% of the DMD patients results in lower global IQ performance scores, has been attributed mostly to deficits in the verbal IQ, language and learning ability [START_REF] Billard | Cognitive functions in Duchenne muscular dystrophy: a reappraisal and comparison with spinal muscular atrophy[END_REF][START_REF] Billard | Reading ability and processing in Duchenne muscular dystrophy and spinal muscular atrophy[END_REF][START_REF] Ogasawara | Downward shift in IQ in persons with Duchenne muscular dystrophy compared to those with spinal muscular atrophy[END_REF]. Structural changes have been observed in some DMD patients like cerebral atrophy [START_REF] Yoshioka | Central nervous system involvement in progressive muscular dystrophy[END_REF], neuronal loss, and gliosis [START_REF] Jagadha | Brain morphology in Duchenne muscular dystrophy: a Golgi study[END_REF].

Most of our understanding of the function of dystrophin in the brain is derived from studies done on mdx mice, which express all the small dystrophin isoforms including Dp71, but lack the full-length dystrophin. The behavioral and cognitive changes observed in mdx mice include impaired memory retention, spatial memory and procedural learning [START_REF] Muntoni | Passive avoidance behaviour deficit in the mdx mouse[END_REF][START_REF] Vaillend | Influence of dystrophin-gene mutation on mdx mouse behavior. I. Retention deficits at long delays in spontaneous alternation and bar-pressing tasks[END_REF][START_REF] Vaillend | Impaired long-term spatial and recognition memory and enhanced CA1 hippocampal LTP in the dystrophin-deficient Dmd(mdx) mouse[END_REF]. Interestingly, the behavioral abnormalities in mdx mice normalize upon restoration of dystrophin suggesting reversal of neuronal function [START_REF] Goyenvalle | Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers[END_REF].

In the brain, normally, the full-length dystrophin is transcribed from the Brain-and Purkinje cell promoters [START_REF] Lidov | Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons[END_REF]. Dp427 and DAPC-like complexes are expressed at postsynapse of neurons in the hippocampus, cerebellum and cerebral cortex. Altered synaptic plasticity has been observed in hippocampal neurons and in Purkinje cells of mdx mice that lack only the Dp427 isoform [START_REF] Anderson | Long-term depression is reduced in cerebellar Purkinje cells of dystrophindeficient mdx mice[END_REF][START_REF] Anderson | Enhanced homosynaptic LTD in cerebellar Purkinje cells of the dystrophic MDX mouse[END_REF][START_REF] Mehler | Enhanced sensitivity of hippocampal pyramidal neurons from mdx mice to hypoxia-induced loss of synaptic transmission[END_REF][START_REF] Vaillend | Spatial discrimination learning and CA1 hippocampal synaptic plasticity in mdx and mdx3cv mice lacking dystrophin gene products[END_REF][START_REF] Vaillend | Facilitated NMDA receptor-mediated synaptic plasticity in the hippocampal CA1 area of dystrophin-deficient mice[END_REF]. Authors have suggested that dystrophin would be involved in the clustering of subpopulations of γ-aminobutyric acid receptors (GABAA-R). γ-Aminobutyric acid is the major inhibitory neurotransmitter in the nervous system suggesting a role of dystrophin at inhibitory synapses. Co-localization between dystrophin and the α2 subunit of GABAA-R was also shown in cultured hippocampal neurons in vitro [START_REF] Brünig | GABAergic terminals are required for postsynaptic clustering of dystrophin but not of GABA(A) receptors and gephyrin[END_REF]. Moreover loss of dystrophin and/or dystrobrevin leads to severe reduction in the number and size of GABAA-R [START_REF] Grady | Role for alpha-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies[END_REF][START_REF] Kneussel | Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice[END_REF][START_REF] Sekiguchi | A deficit of brain dystrophin impairs specific amygdala GABAergic transmission and enhances defensive behaviour in mice[END_REF]. Although functional molecular data are lacking about the interaction between dystrophin and GABAA-R, electrophysiological studies support this hypothesis [START_REF] Anderson | Altered inhibitory input to Purkinje cells of dystrophin-deficient mice[END_REF][START_REF] Kueh | GABAA RECEPTOR EXPRESSION AND INHIBITORY POST-SYNAPTIC CURRENTS IN CEREBELLAR PURKINJE CELLS IN DYSTROPHIN-DEFICIENT mdx MICE[END_REF][START_REF] Vaillend | Facilitated CA1 hippocampal synaptic plasticity in dystrophin-deficient mice: role for GABAA receptors?[END_REF].

Additional studies are necessary to better characterize and analyze the role of dystrophin at the synapse and its contribution to cerebral network activity. Beyond a direct involvement of dystrophin in neuronal activity, authors suggested a function of full-length dystrophin in the formation and maintenance of blood brain barrier [START_REF] Nico | Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice[END_REF][START_REF] Nico | Altered blood-brain barrier development in dystrophic MDX mice[END_REF]. The Dp140 isoform is manly expressed during development, but expression in the microvasculature of the adult brain was suggested as well. Dp71 is expressed in glia cells, notably in perivascular astrocytes and the Bergmann glia in the cerebellum surrounding the endothelial cells [START_REF] Blake | Different Dystrophin-like Complexes Are Expressed in Neurons and Glia[END_REF][START_REF] Lidov | Dp140: a novel 140 kDa CNS transcript from the dystrophin locus[END_REF]. The distinct expression of Dp71 at the glial-vascular interface observed in humans, as well as in animal models, suggested a role of Dp71 in blood brain barrier function. It has been reported that Dp71 is involved in regulation of water homeostasis via the AQP4 water permeable channel and is involved in potassium (K + ) buffering mediated by the inward rectifier K + channel (Kir4.1) [START_REF] Connors | The potassium channel Kir4.1 associates with the dystrophinglycoprotein complex via alpha-syntrophin in glia[END_REF][START_REF] Nicchia | Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complex[END_REF]. Additionally, Dp71-null mice display cognitive defects that were linked to excitatory synapse organization and function. Hence, another role for Dp71 was proposed at excitatory glutamatergic synapses that was supported by some experimental evidence [START_REF] Daoud | Analysis of Dp71 contribution in the severity of mental retardation through comparison of Duchenne and Becker patients differing by mutation consequences on Dp71 expression[END_REF]. Further studies need to be done to elucidate the precise functions of Dp71 and its alternative splicing variants.

Dystrophin and the retina

In the retina following dystrophin isoforms are expressed: Dp427m, Dp260, Dp140, and Dp71 [START_REF] Schmitz | Dystrophin in the retina[END_REF]. In the mouse retina, the full-length and Dp260 dystrophin isoforms are found at the outer plexiform layer (OPL), a region where the highly specialized synapses between the glutamatergic photoreceptor with horizontal and with bipolar cells are located [START_REF] Dalloz | Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina[END_REF][START_REF] Pillers | Dystrophin expression in the human retina is required for normal function as defined by electroretinography[END_REF][START_REF] Schmitz | Dystrophin in the retina[END_REF]. The dystrophin isoforms in this region are associated with the photoreceptor terminals and suggest involvement of dystrophin in synaptic transmission (Ueda et al., 1997), but the precise mechanisms still remain to be elucidated. The short Dp71 dystrophin isoform is present in astrocytes and in the principal glial cells of the retina, the Müller cells, which are located at the inner limiting membrane that is the basal lamina separating the neural retina from the vitreous humour. Like in the brain, Dp71 expression in these cells is limited to the glialendfeet. Transgenic mice lacking all retinal isoforms as well as Dp71 null mice exhibit a mislocalization of the Kir4.1 potassium channel and the AQP4 channel with reduced AQP4 levels. Thus, like in the brain, a role of Dp71 in the maintenance of potassium and water homeostasis as well as in the regulation of retinal vascular permeability has been proposed [START_REF] Dalloz | Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina[END_REF][START_REF] Sene | Functional implication of Dp71 in osmoregulation and vascular permeability of the retina[END_REF][START_REF] Wersinger | Reevaluation of Dystrophin Localization in the Mouse Retina[END_REF].

The best characterized non-muscle manifestation of DMD is described in the visual system. In 80% of the patients, there is a reduction in the amplitude of the b-wave response revealed by the dark-adapted (scotopic) electroretinogram (ERG). The ERG measures electric responses of the various cell types in the retina upon light stimulation. Depending on the kind of stimuli and stimuli adaptation different patterns can be recorded reflecting the activity of different cell types in the retina. Usually dark adapted response reflects the activity of the rod photoreceptors system. The abnormal b-wave in DMD patients and in mdx mice, that lack all dystrophin isoforms, reflexts changes of synaptic transmission between the rod photoreceptors and the ON-bipolar cells [START_REF] Cibis | The effects of dystrophin gene mutations on the ERG in mice and humans[END_REF][START_REF] Pillers | Dystrophin expression in the human retina is required for normal function as defined by electroretinography[END_REF]. There is a correlation between the type of mutation, the affection of the respective DMD gene product and the presence of an abnormal ERG response. In patients, in whom the mutation does not abolish the expression of the Dp260 isoform have normal ERGs, while most C-terminally located mutations lead to changes in the b-wave. These findings suggest that Dp260 retinal isoform indeed plays a role in the b-wave generation [START_REF] Dalloz | Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina[END_REF]. In dystrophic mdx 3cv mice that lack all short dystrophin isoforms due to a mutation near the 3' end of Dmd show similar abnormalities in b-wave amplitude, further indicting the requirement of specific dystrophin isoforms for normal retinal function [START_REF] Souza | A novel dystrophin isoform is required for normal retinal electrophysiology[END_REF]. It is noteworthy, however, that, unlike in humans, all three isoforms need to be abolished in order to provoke the ERG changes in mice [START_REF] Souza | A novel dystrophin isoform is required for normal retinal electrophysiology[END_REF]. A similar phenotype was observed in dystroglycan deficient mice suggesting a joint role of dystrophin and its DAPC-like complex (Satz et al., 2009). In addition, authors recently reported that some DMD patients, with mutations affecting the Dp260 isoform, exhibites a deficit in red-green discrimination [START_REF] Costa | Red-Green Color Vision Impairment in Duchenne Muscular Dystrophy[END_REF].

The role of dystrophin in satellite cells

A recent study done by Dumont and colleagues described for the first time a functional role for dystrophin in the muscle stem cells [START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF]. It was generally assumed that dystrophin would not be expressed in satellite cells. However, the study showed expression of full-length dystrophin in activated satellite cells and provided data for the functional role of dystrophin in for the establishment of polarity through interaction with the microtubuleassociating protein (MAP)/microtubule affinity-regulating kinase 2 (Mark2). During apicobasal asymmetric division satellite cells give rise to two daughter cells with a distinct cell fate.

The one daughter cell differentiates and contributes to the myogenic progenitor cells, which fuse with the myofiber for growth and repair, while the other daughter cell keeps its stem cell potential. During this process the expression of dystrophin was confined to the cell that maintained the satellite cell state. The novel role for dystrophin has a direct impact on the pathophysiological understanding of dystrophin deficient conditions. The authors of the study presented data showing the impaired polarity establishment and loss of asymmetric cell division in dystrophin deficient mdx mice due to dysregulation of mitotic spindle orientation.

Subsequently, the animals had reduced numbers myogenic progenitors, which could explain the impaired regeneration observed in dystrophic conditions despite the high number of satellite cells normally observed in mdx mice and DMD patients. This novel function of dystrophin opens a new avenue of research of dystrophinopathies. The defects observed in asymmetric stem cell division in dystrophic mice now allow for a re-interpretation of the muscular deficits observed in DMD patients [START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF].

Mutations of the DMD gene

Thousands of independent mutations have been described in the DMD gene that has a high spontaneous mutation rate of 10 -4 gene/generation [START_REF] Blake | Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle[END_REF]. The "reading frame rule" explains why neither the size nor the position of the DMD deletion/mutation shows a good correlation with the phenotype (genotype-phenotype relation). It postulates, in case a deletion disrupts the open DMD reading frame, the resulting premature termination of translation prevents the proper expression of dystrophin. The nearly total absence of dystrophin causes the severe Duchenne variant of muscular dystrophy. In cases, where the deletion does not disrupt the reading frame, dystrophin can be produced albeit in a truncated form that consenses only partial function. These mutations tend to cause the milder Becker variant of muscular dystrophy [START_REF] Monaco | An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus[END_REF].

The mutations found in DMD patients and their corresponding phenotype have vastly contributed to the understanding of the function of specific dystrophin domains and have facilitated the development of mini-or micro-dystrophins that are tested for so-called gene therapeutic interventions [START_REF] Blake | Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle[END_REF].

Large deletions are the most common mutations in DMD or BMD and are identified in approximately 65% of the patients. Two mutational hotspots spanning exons 45-53 and exons 2-20 have been identified [START_REF] Koenig | The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion[END_REF][START_REF] Koenig | Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility[END_REF]. Small deletions and point mutations have been observed in one third of the patients, most commonly leading to premature stop codons [START_REF] Lenk | Point mutations at the carboxy terminus of the human dystrophin gene: implications for an association with mental retardation in DMD patients[END_REF][START_REF] Roberts | Dystrophins in Vertebrates and Invertebrates[END_REF].

The rod domain has been found to accommodate large in-frame deletions resulting in a mild Becker type dystrophy. The rod domain acts as a bridge or spacer between the functionally important N-terminal actin binding and the cysteine-rich and C-terminal domain. Skipping this region results in truncated dystrophin protein isoforms that seem to function normally [START_REF] England | Very mild muscular dystrophy associated with the deletion of 46% of dystrophin[END_REF].

Revertant fibers in DMD patients and in mdx mice

Mutations in DMD patients and in the mdx mouse disrupt the open reading frame and prevent the expression of dystrophin. Nevertheless, the emergence of dystrophin positive fibers, the so-called "revertant fibers" (RFs), in an otherwise dystrophin negative background has been observed [START_REF] Arechavala-Gomeza | Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: implication for clinical trials[END_REF][START_REF] Danko | The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy[END_REF][START_REF] Hoffman | Somatic reversion/suppression of the mouse mdx phenotype in vivo[END_REF][START_REF] Nicholson | The 'rescue' of dystrophin synthesis in boys with Duchenne muscular dystrophy[END_REF][START_REF] Nicholson | Dystrophin in skeletal muscle. II. Immunoreactivity in patients with Xp21 muscular dystrophy[END_REF]S.D. Wilton et al., 1997). RFs express an internally truncated form of dystrophin. This suggests that secondary events must have occurred that resulted in the re-establishment of the reading frame. In the mdx mouse, RFs have been studied more intensely, and it has been shown that they emerge around birth in short segments and increase in size and number with age. The revertant event is thought to take place in the muscle precursor cells that are then activated and proliferate during the repetitive cycles of de-and regeneration that are characteristic for the dystrophic muscle.

This process leads to the expansion of RFs [START_REF] Lu | Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion[END_REF][START_REF] Yokota | Expansion of revertant fibers in dystrophic mdx muscles reflects activity of muscle precursor cells and serves as an index of muscle regeneration[END_REF]. RFs form clonal clusters but exhibit Dmd splicing pattern, which differs between neighboring independent clusters. The different dystrophin isoforms in the individual clusters have been confirmed on the protein level using exon-specific antibodies [START_REF] Lu | Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion[END_REF][START_REF] Thanh | Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin[END_REF], as well as on the mRNA level by RT-PCR (S. D. Wilton et al., 1997). On the mRNA level, such shortened transcripts have also been observed in the normal muscle tissue of the mouse, but whether they are translated into protein has not yet been shown. The size of each cluster can encompass up to 100 fibers and reach 100 mm length at 18 months of age [START_REF] Lu | Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion[END_REF].

Such RFs can also be found in muscles of other breeds of mdx mice that bear different mutations [START_REF] Danko | The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy[END_REF][START_REF] Echigoya | Mutation Types and Aging Differently Affect Revertant Fiber Expansion in Dystrophic Mdx and Mdx52 Mice[END_REF] as well as in the Golden Retriever Dystrophic Dog (GRMD) [START_REF] Schatzberg | Alternative dystrophin gene transcripts in golden retriever muscular dystrophy[END_REF]. In human DMD patients the percentage of RFs ranges between 0.01 and 7 percent and, in contrast to the mdx mice, no RF expansion with age has been observed. A correlation between the emergence of RFs and an improvement of DMD symptoms has not been observed so far [START_REF] Arechavala-Gomeza | Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: implication for clinical trials[END_REF]. The amount of RFs is not only influenced by the location of the mutation, but also by the genetic background of the mouse [START_REF] Echigoya | Mutation Types and Aging Differently Affect Revertant Fiber Expansion in Dystrophic Mdx and Mdx52 Mice[END_REF]. More recent studies, which support the hypothesis of clonal expansion of RFs, suggest the occurrence of de novo reversions throughout the whole lifespan and show that such events could take place in the heart as well [START_REF] Pigozzo | Revertant Fibers in the mdx Murine Model of Duchenne Muscular Dystrophy: An Age-and Muscle-Related Reappraisal[END_REF].

The natural emergence of RFs in DMD patients has to be taken into account when assessing the efficacy of (gene) therapeutic approaches. On the other hand, it is important to understand the underlying molecular mechanisms and the origin and expansion of RFs as this phenomenon could be exploited for novel therapies and will contribute to the better understanding of the disease. Furthermore, the natural phenomenon of RFs provides the basis for one of the most promising therapeutic approaches against DMD, the exon skipping which will be explained in the next section.

Therapies for DMD

Different therapies against DMD exist and are being continuously developed. The aim is to slow the progression of the disease by immune modulation (e.g. via steroids and inhibitors of transforming growth factor), by inducing or introducing proteins that may compensate for dystrophin deficiency in the myofiber (e.g. utrophin, biglycan, and laminin), or enhance the regenerative response of the muscle (e.g. myostatin and activin 2B). Alternatively, studies are on the way that aim to introduce a more functional gene into the patient using viral gene transfer [START_REF] Koppanati | Improvement of the mdx mouse dystrophic phenotype by systemic in utero AAV8 delivery of a minidystrophin gene[END_REF][START_REF] Kornegay | Widespread Muscle Expression of an AAV9 Human Mini-dystrophin Vector After Intravenous Injection in Neonatal Dystrophin-deficient Dogs[END_REF] or to repair the DMD gene of the patient. Due to a multitude of technical and biological obstacles, however, the advance of gene therapies and of cell transplantation into the clinic is very slow despite extensive research [START_REF] Maffioletti | Stem cell transplantation for muscular dystrophy: the challenge of immune response[END_REF][START_REF] Seto | Gene replacement therapies for Duchenne muscular dystrophy using adeno-associated viral vectors[END_REF][START_REF] Tremblay | A Case for Immunosuppression for Myoblast Transplantation in Duchenne Muscular Dystrophy[END_REF][START_REF] Wang | Immunity and AAV-Mediated Gene Therapy for Muscular Dystrophies in Large Animal Models and Human Trials[END_REF].

A more promising therapy against DMD has been developed using small molecules, artificial antisense oligonucleotides (AONs) that "repair" the open reading frame of the mutated gene through skipping of exons, which flank the original mutation. If this would be achieved in a highly predictive way, the reading frame could be restored and such transcript translated into a truncated but partially functional dystrophin [START_REF] Dunckley | Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides[END_REF][START_REF] Goyenvalle | Therapeutic approaches to muscular dystrophy[END_REF][START_REF] Matsuo | Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe[END_REF][START_REF] Yokota | Optimizing exon skipping therapies for DMD[END_REF]. This so-called "exon skipping" approach is similar to the phenomenon of RFs, in which exon skipping occurs "naturally" as described in the previous section. Alternatively, exon skipping can be achieved using specific mRNA splicing molecules (U7 or U1 RNA) which splice out extra exons. A disadvantage of the U7 or U1 RNA molecules as opposed to AONs is the delivery system which uses viral vectors and could provoke an immune response. [START_REF] Goyenvalle | Rescue of Dystrophic Muscle Through U7 snRNA-Mediated Exon Skipping[END_REF][START_REF] Goyenvalle | Enhanced Exon-skipping Induced by U7 snRNA Carrying a Splicing Silencer Sequence: Promising Tool for DMD Therapy[END_REF][START_REF] Lorain | Transient immunomodulation allows repeated injections of AAV1 and correction of muscular dystrophy in multiple muscles[END_REF].

Exon skipping via AONs

AONs used in exon skipping recognize a specific exon-intron boundary of the pre-mRNA, result in modulation of the splicing pattern and aim to restore the dystrophin expression. The mutated exon and, if necessary, some of its neighboring exons are thus remioved and the reading frame of the pre-mRNA is reconstituted, albeit shorter. In pre-clinical studies for DMD, the most promising candidate drugs have proven to be the 2'-O-methyl phosphorothioate (2'OMe-PS) and the phosphordiamidate morpholino oligomers (PMO), both targeting exon 51 [START_REF] Aoki | Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery[END_REF][START_REF] Heemskerk | In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping[END_REF][START_REF] Pramono | Induction of Exon Skipping of the Dystrophin Transcript in Lymphoblastoid Cells by Transfecting an Antisense Oligodeoxynucleotide Complementary to an Exon Recognition Sequence[END_REF][START_REF] Yokota | Efficacy of systemic morpholino exonskipping in Duchenne dystrophy dogs[END_REF]. These two compounds entered phase 2/3 clinical trials. However both compounds showed little clinical efficacy likely as the trial duration was too short in order to discriminate a clear clinical improvement [START_REF] Cirak | Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study[END_REF][START_REF] Goemans | Systemic administration of PRO051 in Duchenne's muscular dystrophy[END_REF][START_REF] Mendell | Eteplirsen for the treatment of Duchenne muscular dystrophy[END_REF][START_REF] Voit | Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study[END_REF].

A great disadvantage of these two molecules had already been observed during preclinical studies because they were not effective in treating cardiac and the central nervous symptoms.

Recently, a novel promising AONs have been developed that overcome these limitations.

Peptide-conjugated PMOs offers significantly higher efficiency than PMOs. In animal models, it has been shown that the peptide-conjugated PMOs succefully restored dystrophin expression and improve the function of skeletal and cardiac muscles [START_REF] Wu | Long-term rescue of dystrophin expression and improvement in muscle pathology and function in dystrophic mdx mice by peptide-conjugated morpholino[END_REF].

Moreover, an important advance in the treatment of the multi-systemic DMD, Goyenvalle and colleagues used tricycle-DNA oligomers in mdx and utrophin/dystrophin double knockout mice and clearly demonstrated dystrophin rescue in skeletal, heart and to some extent in the brain after systemic delivery of the drug [START_REF] Goyenvalle | Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers[END_REF]. Functional tests have confirmed the clinical improvement in the treated animals.

Currently the mdx and mdx52 mice are the most widely used models for testing the efficacy of AONs or other splicing modulators [START_REF] Aoki | Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery[END_REF][START_REF] Lu | Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse[END_REF]. Mdx52 bears a deletion of exon 52, which is one of the mutational hotspots in DMD patients. The mdx52 mouse and can be used to test AONs sequences for skipping exon 51 and 53. [START_REF] Aoki | Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery[END_REF]. The use of animal models to test different AONs and assess their main properties like stability, efficacy and toxicity [START_REF] Yokota | Efficacy of systemic morpholino exonskipping in Duchenne dystrophy dogs[END_REF] is indispensable. Pre-clinical evidence of the therapeutic potential of AONs have been provided by numerous in vivo studies [START_REF] Aoki | Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery[END_REF][START_REF] Fletcher | Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide[END_REF][START_REF] Fletcher | Morpholino Oligomer-Mediated Exon Skipping Averts the Onset of Dystrophic Pathology in the mdx Mouse[END_REF][START_REF] Gebski | Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle[END_REF][START_REF] Lu | Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse[END_REF][START_REF] Yokota | Efficacy of systemic morpholino exonskipping in Duchenne dystrophy dogs[END_REF].

Aims of the study

Dystrophin is a protein expressed in all types of muscle as well as in non-muscle tissue [START_REF] Hoffman | Dystrophin: The protein product of the duchenne muscular dystrophy locus[END_REF][START_REF] Hoffman | Cell and fiber-type distribution of dystrophin[END_REF][START_REF] Uchino | Localisation and characterisation of dystrophin in the central nervous system of controls and patients with Duchenne muscular dystrophy[END_REF]. It was discovered almost 30 years ago as the protein missing in Duchenne muscular dystrophy, a severe X-linked disorder known for more than 100 years [START_REF] Duchenne | Recherches sur la paralysie musculaire pseudohypertrophique ou paralysie myo-sclérosique[END_REF][START_REF] Koenig | Complete cloning of the duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF]Anthony P. Monaco et al., 1986).

Dystrophin is a rod-shaped cytoplasmic protein that physically links the cytoskeleton to the extracellular matrix. The identification of dystrophin close to the inner surface of the plasmalemma pushed the research efforts toward understanding the physiological role of dystrophin in the cell membrane. Vast progress has been made in the last two decades in comprehending the role of dystrophin in the muscle. Its protein structure, its binding partners, as well as its coding gene and complex regulatory mechanisms are well characterized.

The advancement of this field was based on studies on DMD patients, and prominent contributions have also been gained through identification and later also generation of animal models. Such models play a tremendously important role in the development of different therapeutic strategies; however, presently DMD cannot be cured. This calls for further investigations into the physiological role of dystrophin, not only in the muscle but also in other organs and tissues where it is expressed. Most of the murine models studying dystrophin are transgenic lines expressing a certain dystrophin isoform or overexpressing mini-or microdystrophins in a particular tissue (Cox et al., 1994b;[START_REF] Gaedigk | Improvement in survival and muscle function in an mdx/utrn(-/-) double mutant mouse using a human retinal dystrophin transgene[END_REF][START_REF] Greenberg | Exogenous Dp71 restores the levels of dystrophin associated proteins but does not alleviate muscle damage in mdx mice[END_REF][START_REF] Hakim | Truncated dystrophins reduce muscle stiffness in the extensor digitorum longus muscle of mdx mice[END_REF][START_REF] Harper | Modular flexibility of dystrophin: Implications for gene therapy of Duchenne muscular dystrophy[END_REF][START_REF] Judge | Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex[END_REF][START_REF] Lai | Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy[END_REF][START_REF] Li | A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy[END_REF][START_REF] Phelps | Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice[END_REF][START_REF] Sakamoto | Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene[END_REF][START_REF] Warner | Expression of Dp260 in muscle tethers the actin cytoskeleton to the dystrophin-glycoprotein complex and partially prevents dystrophy[END_REF].

However, DMD is a multi-system disorder and dystrophin has a high number of isoforms and splicing variants expressed at different cellular and subcellular localizations. This contributes to the pleiotropic function of dystrophin and different effects of gene mutations depending on their location.

Therefore, it would be beneficial to have a more general model that will facilitate the study of dystrophin expression in various tissues in a non-artificial system. In the work that comprises my dissertation, I strove to generate and characterize a Dmd EGFP reporter mouse, which should express a functional dystrophin-EGFP fusion protein.

Aim 1: To insert an EGFP-tag behind dystrophin exon 79 to create a dystrophin reporter mouse

The fluorescently labeled dystrophin protein should be expressed throughout muscle and non-muscle tissues by the natural endogenous promoters, the aim being to target as many dystrophin isoforms as possible. The C-terminus of dystrophin is an important functional domain, known to be expressed in all functional relevant dystrophin isoforms. The last exon 79 of the dystrophin gene is present in most of these isoforms as well as in revertant dystrophin. We thus aimed to insert the EGFP coding sequence behind the last Dmd exon 79, which had to be modified in order for the EGFP sequence to be co-expressed. Thus, we expected to target all full-length dystrophin isoforms (Dp427M, B, P), the retinal specific Dp260 isoform, as well as the shorter Dp140, Dp116, Dp71d and Dp71c isoforms. Only Dp71f and Dp71Δ110 cannot be targeted in our model since alternative splicing of exon 78 excludes also exon 79.

Aim 2: To investigate dystrophin-EGFP expression in different tissues of the mouse

After successful introduction of the EGFP-tag I wanted to investigate the tissue specific expression of the dystrophin-EGFP fusion protein in skeletal, smooth and cardiac tissue as well as in non-muscle tissues like brain and eye. Up to date, no dystrophin reporter mouse model exists and only few models use a fluorescent tag to follow up the expression of dystrophin [START_REF] Bajanca | In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis[END_REF][START_REF] Li | A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy[END_REF][START_REF] Ruf-Zamojski | Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line[END_REF]. Through direct visualization of dystrophin, without the necessity of antibody staining the developed Dmd EGFP model will facilitate the studies of dystrophin and dystrophinopathies ex vivo as well as in vivo.

Aim 3: To exclude the possibility that the C-terminal EGFP-tag causes muscular dystrophy

Adding a C-terminal tag to a protein might hinder its function. Hence, I wanted to analyze the mouse model with respect to possible features of a dystrophinopathy and to check for mis-localization of other components of the dystrophin associated complex (DAPC) that might have been unintentionally caused by the EGFP-tag.

MATERIALS

Chemicals used in the context of this thesis were obtained from AppliChem (Darmstadt, Germany), GE Healthcare (Munich, Germany), Merck AG (Darmstadt, Germany), Roche GmbH (Grenzach-Wyhen, Germany), Carl Roth GmbH (Karlsruhe, Germany) and Sigma Aldrich GmbH (Taufenkirchen, Germany). Media and compounds for cell culture work were obtained from Gibco/ LifeTechnologies (Karlsruhe, Germany), Invitrogen GmbH (Karlsruhe Germany) and VWR (Darmstadt, Germany).

Bacteria and supplies for bacterial culture were obtained from Invitrogen GmbH (Karlsruhe Germany) and Promega (Mannheim, Germany).

Plastic materials

Instruments, equipment

Chemicals

Antibodies

Antibodies were purchased from Abcam (Cambridge, UK), Novocastra/Leica (Wetzlar, Germany) Merck/Millipore (Darmstadt, Germany), Roche (Grenzach-Wyhlen, Germany), DSHB (IO, USA), ThermoFisher Scientific (Wuppertal, Germany). All antibodies are listed in Appendix 10.1, Table A2-3.

Oligonucleotide primers for PCR

All oligonucleotide primers were custom synthesized the the MWG Biotech AG (Ebersberg, Germany) company. A complete list of all used oligonucleotide primers is attached in the Appendix 13.1, Table A1.

Cell lines

Cell lines were cultured in an incubator at 37°C, 95% humidity and 5% CO2 atmosphere. All cell culture work was done under sterile conditions in a biological safety cabinet.

METHODS

Methods for the generation of a transgenic Dystrophin-EGFP reporter mouse

The main aim of this work was to generate a reporter mouse, in which a FLAG-EGFP sequence was inserted behind the last exon of the Dmd gene so that a C-terminally tagged dystrophin-EGFP protein would be expressed. This model will facilitate in vivo studies on dystrophin and DMD, as there would be no need for antibody staining.

In summary, to generate the Dmd reporter allele, I constructed a targeting vector using the method of recombineering so that a 10.943 kb DNA fragment spanning intron 78, exon 79 and part of the 3'UTR of the murine Dmd was excised from a genomic X-chromosomal BAC clone and subcloned. Afterwards, the Dmd exon 79 was modified by changing the stop codon into a leucine and inserting a FLAG-EGFP sequence in-frame, followed by a neomycin cassette Mice were then bred under normal husbandry conditions. The neo cassette was removed by crossing mice from the F1 generation mice with mice that ubiquitously expressed the Cre recombinase [START_REF] Schwenk | A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells[END_REF]. Below I describe the single steps in more detail.

Generation of the targeting vector via recombineering

Generation of the targeting vector was done using the method of recombineering (recombination-mediated genetic engineering) [START_REF] Copeland | MOUSE GENOMIC TECHNOLOGIES: RECOMBINEERING: A POWERFUL NEW TOOL FOR MOUSE FUNCTIONAL GENOMICS[END_REF][START_REF] Liu | A highly efficient recombineering-based method for generating conditional knockout mutations[END_REF][START_REF] Muyrers | Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA[END_REF] Exo encodes a 5'-3' exonuclease that recognizes double strand breaks in linear DNA and digests the 5' end of these DNA strands. The respective 5' overhangs are then bound by the bet encoded protein bringing the homologous regions close together [START_REF] Poteete | What makes the bacteriophage λ Red system useful for genetic engineering: molecular mechanism and biological function[END_REF][START_REF] Stahl | Recombination in phage λ: one geneticist's historical perspective1[END_REF]. The gam encoded protein prevents the degradation of linear DNA via inhibition of the bacterial RecBCD-exonuclease activity. This is necessary as the DNA to be integrated via recombineering is mostly in the form of short PCR fragments, which would be degraded if

RecBCD system was not blocked.

Plasmid DNA preparation from Escherichia coli

Small-scale plasmid DNA preparations for analytical purposes were done using the QIAprep® Spin Miniprep Kit (Qiagen, Hilden, Germany). The method is based on the alkaline lysis of bacterial cells, modified after [START_REF] Birnboim | A rapid alkaline extraction procedure for screening recombinant plasmid DNA[END_REF]. 5 ml of an overnight culture of transformed E. coli JM109 or XL-10 Gold bacteria were used according to manufacturer's instructions.

For preparative purposes midi preparations were performed using HiSpeed Plasmid Midi Kit from the company Qiagen according to manufacturer's instructions with a total bacterial culture volume of 50-100 ml. The isolation and purification is based on the alkaline lysis of bacterial cells and binding of plasmid DNA to anion-exchange columns under low-salt conditions. DNA was eluted in 150 μl TE buffer and concentration adjusted to 1 μg / μl. All plasmid DNA concentrations were determined using a Nanodrop TM 1000 spectrophotometer.

BAC DNA preparation from Escherichia coli

BAC plasmid DNA (clone bMQ389g17, backbone:pBACe3,) encoding a fragment from the Dmd gene of the SV129 mouse was prepared from a glycerol stock after plating single colonies and inoculating a 5 ml overnight culture in LB medium supplemented with chloramphenicol.

Overnight culture was pelleted for 5 min at 2,000 x g, the supernatant removed, the pellet dissolved in 250 µl buffer P1 (from QIAprep® Spin Miniprep Kit from Qiagen) and transferred to a microcentrifuge tube. 250 ml P2 buffer were added, followed by mixing and incubation for <5 min at room temperature. Buffer N3 was added, followed by mixing and incubation on ice for 5 min. The supernatant was cleared by two rounds of centrifugation at 13,600 x g for 5 min in a tabletop centrifuge. Each time the supernatant was transferred into a new tube.

DNA was precipitated by adding 750 µl isopropanol, mixing and centrifugation for 20 min at 13,600 x g. The pellet was washed once in 70% ethanol, air-dried and dissolved in 50 μl HPLCwater ('NCI at Frederick: Protocols'). The BAC DNA was analyzed for containing the correct Dmd genomic sequence using specific PCR primers that amplified a fragment at around Dmd exon 79. BAC DNA was used for recombineering.

Transformation of Escherichia coli with plasmid DNA

Chemically competent E. coli JM109 and XL10-Gold bacteria were used for transformation with plasmid DNA. The competent bacteria were thawed on ice and aliquots of 50 µl or 100 µl, respectively, were used for each transformation. Plasmid DNA or ligation mixture was added to the cells followed by 30 min incubation on ice. Cells were heat shocked for 2 minutes at 42°C in water bath and put back on ice for 2-3 minutes. The fast change in the temperature leads to stretching of the bacterial membrane, which supports the plasmid uptake.

Subsequently, 500 µl pre-warmed SOC -medium (SOB medium plus 20 mM glucose; SOB medium: 0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 20 mM MgSO4) were added to the cells and the samples were incubated for at least 45 min in the shaker at 37°C. The bacteria were pelleted at 5,000 x g for 2 min and 450 µl medium was discarded. The cells were resuspended in the remaining volume and plated on LB plates supplemented with either ampicillin (100 µg/ml), kanamycin (30 µg/ml), or chloramphenicol (20 µg/ml) for later selection of positive clones transformed with plasmid or BAC DNA, encoding the respective resistance gene. Plates were incubated over night at 37°C.

Culturing conditions and long-term storage of Escherichia coli

Single bacterial clones were picked from the plates and used to inoculate a culture with 2-150 ml LB-medium supplemented with the respective antibiotic. Agar plates with transformed bacteria were stored at 4°C for several weeks. For long term storage glycerol stocks were prepared by adding glycerol to the E. coli overnight culture to a final concentration of 20%.

The mixture was flash frozen in liquid nitrogen and stored at -80°C. Bacteria were recovered by scratching a little amount out of the glycerol stock and plating it on LB agar plates containing appropriate antibiotics. Single positive clones were picked and inoculated into a bacterial culture for small or bigger scale DNA plasmid preparation.

Preparation of electro-competent cells for recombineering and induction of λ-genes

Electro-competent cells for recombineering were prepared by inoculation of 0.5 ml of an overnight culture (E. coli SW106) in 35 ml LB medium. The bacterial cells were incubated at 32°C until they reached an OD600 of 0.4-0.6. For activation of the recombineering enzymes the bacterial culture was incubated for 15 min at 42°C in a shaking water bath. Half of the culture was induced; the other half was left at 32°C and used as a control. Both the induced and uninduced cells were rapidly chilled on ice/ -water slurry and incubated for 10 min on ice.

Cells were spun down for 5 min at 5,300 x g and washed three times with pyrogen free water.

The supernatant was transferred into microcentrifuge tubes and centrifuged at 10,000 x g for 30 sec at 4°C. The supernatant was aspirated carefully and cells suspended in 1 ml of ice-cold H2O. After repeating this step, the cell pellet was resuspended in 200 µl of sterile cold distilled H2O and kept on ice until used.

For expression of the Cre-gene and subsequent excision of the selectable marker (neo cassette), 0.5 ml of overnight E. coli SW106 culture were incubated in 35 ml of LB medium at 32°C until reaching OD600=0.4. Subsequently, arabinose was added to a final concentration of 0.1% (w/v) and the cells were incubated for a further 1 h at 32°C. Cells were then used for electroporation.

Electroporation of competent bacterial cells

For the introduction of DNA into electro-competent cells via a mixture of bacteria and plasmid-DNA was poured into a pre-cooled electroporation cuvette (width: 0.1 cm) and electroporation was carried out with following parameters: voltage: 1.65 kV, capacity: 25 µF, resistance: 200 Ω. The mixture was then transferred to and resuspended in 1 ml SOC medium, incubated for 1 h at 32°C and streaked onto LB plates without antibiotics (recombineering) or containing appropriate antibiotics (electroporation of retrieval vector or BAC DNA). For the introduction of the Dmd fragment from the BAC DNA into pCR-Script and generation of the Dmd exon 79 retrieval vector, 100 ng linear plasmid DNA, and 100 ng BAC DNA were added to 50 μl heat-inducted electro-competent cells. For the generation of the targeting vector, electroporation was done using 10 ng retrieval vector and 150 ng linearized PL452 vector containing the modified Dmd exon79 with the EGFP-FLAG-loxP flanked neo cassette. For the excision of the selection cassette, 1 ng plasmid DNA was added to 50 μl arabinose induced electro-competent cells. Correct recombinants and positive clones were then picked and analyzed via colony PCR and automatic sequencing.

Polymerase chain reaction (PCR)

The PCR is a method for the exponential amplification of DNA fragments. In the first step (denaturation), the DNA is separated into single strands, followed by the annealing process, at which oligonucleotide primers specific for a particular DNA region bind at the single Cycling conditions for the PCR reactions were as follows: 95°C, 5 min; 25-35x (95°C, 30-60 sec;

54-65°C, 30-60 sec; 72°C for the duration of 1 min/kb); 72°C, 10 min.

The GoTaq TM DNA-Polymerase was used for colony PCR to find and verify correctly cloned plasmids in the positive bacterial clones. Clones were picked and diluted in 20 µl water, boiled and 1 µl was used as template for the PCR reaction.

Oligonucleotide primers used for cloning contained 5' of the primer binding sequence overhangs with the characteristic recognition sequence for the restriction endonucleases which allowed the digestion of the linear fragments and their subsequent cloning into a vector. They were specifically HPLC purified to prevent the incorporation of falsely synthesized nucleotides. The modified exon 79 fused with the FLAG sequence was also incorporated into the oligonucleotide via primer overhangs.

DNA digestion with restriction enzymes

For restriction analysis plasmid DNA or BAC DNA were digested with restriction endonucleases obtained from New England Biolabs and used in the recommended buffers in a 30 μl reaction volume. For 1 µg DNA 2-10 units restriction enzymes and buffer were used and incubated for 1 to 3 hours at 37°C. Correct digestion was verified by agarose gel electrophoresis (see section 3.1.10). For subcloning of the 10.943 kb fragment from BAC DNA into pCR-Script I used the restriction endonucleases SalI, HindIII and NotI. For cloning of FLAG-EGFP-loxP-neo-loxP into PL452 I used the restriction endonucleases KpnI, EcoRI, BamHI, SacII.

Linearization of the retrieval plasmid and of the targeting vector was done by HindIII and SalI digestion, respectively. BamHI and BglII control digestions of the targeting vector were performed as these two enzymes were to be used for Southern blot analysis of successfully transformed ES cell clones after positive selection. For the cloning of the modified Dmd exon 79 plus FLAG-mEGFP cassette into pCMV-Cox8 vector PstI and HindIII were used.

Agarose gel electrophoresis

Agarose gel electrophoresis separates linear DNA fragments according to their size. 0.7-1.5%

(w/v) agarose gels in 0.5 x TBE buffer (5x TBE stock solution: 450 mM Tris Base, 450 mM Boric acid, 10 mM EDTA, add 1 L H20) was used. The gels were prepared by dissolving the required amount of agarose in TBE buffer through boiling in a microwave stove. After the solution had cooled down, ethidium bromide was added to the agarose solution to a final concentration [0.3 μg/ml] immediately before casting the gel. Ethidium bromide fluoresces under UV light when intercalating with DNA, hence enabling its detection. Solidified agarose gels were placed into the gel chamber, the DNA samples were mixed with 6 x DNA loading dye (0.25% bromphenol blue; 15% Ficoll 400) and pipetted into the gel pockets. In parallel, a standard molecular size marker was loaded for determination of the fragment lengths. Electrophoresis was performed at 8-12 V/cm in 0.5% TBE buffer. DNA bands were visualized with a transilluminator (λ = 366 nm) and documented with a camera (Gel Doc, Bio-Rad, Munich).

DNA extraction from agarose gels

For the extraction and purification of DNA fragments of interest from an agarose gel, these were visualized on the gel by UV-light (λ = 366 nm) and the gel piece containing the fragment was cut out with a scalpel. The purification was done using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. The elution of the DNA fragment was done with 30 µl TE buffer.

Purification of the PCR products

PCR products used for cloning were purified after digestion with restriction enzymes in order to remove excessive reaction components and adhering endonucleases. The purification was performed using the Qiagen PCR Purification Kit (Qiagen) according to manufacturer's instructions. Elution volume was 30 µl.

Cloning of DNA Fragments

Digested and purified PCR products and agarose gel fragments were used for cloning into a plasmid that was digested and linearized with the same restriction enzymes as the fragment to be inserted in order to produce compatible ends. The recipient linearized plasmid DNA (backbone) and the DNA fragment to be introduced into the vector (insert) were used in a ligation reaction at a molar ratio of 3:1 (insert:backbone). The molecular mass of each fragment was calculated using the following formula:

insert mass [ng] = 3 x (insert length [bp]/ backbone length [bp]) x vector mass [ng].
In a typical reaction, 20 μl ligation reaction 50 ng backbone vector were used, together with 2x Quickligase® buffer and Quickligase® enzyme (Invitrogen) for 20 min at room temperature.

1-5 μl ligation mix was used for transformation of chemically competent bacterial cells (section 6.2.3).

For the Southern blot analysis, 5' and 3' DNA probes were generated for the identification of positive ES clones that had underwent homologous recombination with the targeted region at the murine Dmd locus. The fragments were subcloned into the pGEM-T easy vector (Promega) via a process called "TA cloning". The vector contains 5' T-overhangs. Blunt end PCR-Fragments that were generated with the proof-reading Phusion TM DNA-polymerase (Finnzymes, Espoo, Finland) were incubated for 12 min at 72°C with the GoTaq TM DNA-Polymerase (Promega, Mannheim, Germany) and dATPs in order to add 3' A-overhangs to the fragments for subsequent TA cloning. Ligation and transformation were carried out as described above.

Positive clones were identified using colony PCR, DNA was then purified from the bacteria by mini or midi-preparation as described.

DNA sequencing

For each newly cloned DNA fragment, the correct sequence was verified via Sanger sequencing [START_REF] Sanger | DNA sequencing with chain-terminating inhibitors[END_REF]. The method is based on the use of fluorescently marked didesoxynucleotides in a reaction that leads to a base specific chain termination during DNA-synthesis. This leads to generation of DNA fragments of different lengths, which can be separated by various electrophoretic techniques (e.g. capillary electrophoresis) and assigned to the corresponding di-desoxynucleotide. In this way, the nucleotide sequence of a DNA fragment can be determined precisely up to a read length of ≈800-1,000 bp. Automatic sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's instructions and an ABI 3100 Genetic Analyzer (Applied Biosystems). For each sequencing reaction, 150 ng of plasmid were used. The sequence reads were analyzed using the DNAStar software.

General tissue culture methods

Tissue culture conditions for eukaryotic cell lines

Eukaryotic cell lines and primary, isolated cells were kept in a cell culture incubator at 37°C, 5% CO2 and 80-90% humidity. The cells were supplemented every 2 to 3 days with fresh medium. The adherent 293 and COS1 cell lines used in this work were cultured in DMEM supplemented with 10% (v/v) fetal calf serum, and 1% penicillin/ streptomycin.

Cells were split at approximately 90% confluence every 3-4 days by trypsination according to the following protocol: Cells were washed once with PBS to remove residual FCS, which would interfere with trypsin activity. After washing, trypsin-EDTA solution (2 ml for 25 cm 2 dishes, 3 ml for 75 cm 2 dishes, and 5 ml for 175 cm 2 dishes) was added. After 1 min, trypsin was removed and residual enzyme was inactivated by addition of an excess DMEM supplemented with FCS. If necessary, the culture flask had to be incubated sometimes for 3-5 min at 37°C until all cells detached. Cells were than resuspended, and eventually split. The isolation and culturing of single fibers with their attached satellite cells is described in section 6.5.3.. All cell cultures were regularly tested for potential mycoplasma contamination using the VenorGEM® Mycoplasma Diagnosis test (Biochrom).

Freezing and thawing of cells

For long-term storage, cells were kept in liquid nitrogen. Adherent cells were trypsinized, diluted in excess of complete culture medium and counted. The cell suspensions were then transferred to a 50 ml Falcon tube and pelleted by centrifugation for 10 min at 200 x g. The supernatants were discarded and the cell pellets were resuspended in 90% FCS, 10% DMSO at a concentration of 10 7 cells/ml. 1 ml aliquots of the cell suspensions were transferred to cryotubes in a pre-cooled (4°C) freezing container and were incubated at -80°C overnight before the cryotubes were stored in liquid nitrogen for permanent storage.

For restauration of cryo-stocks, cells were thawed rapidly in a water bath at 37°C. The cell suspension was diluted in 10 ml pre-warmed medium in a 15 ml Falcon tube and centrifuged for 5 minutes at 200 x g. The DMSO-containing supernatant was discarded and the cell pellet was resuspended using 12 ml of complete medium. Finally, the cell suspension was transferred into a 75 cm 2 cell culture flask and cultured at 37°C. F is the dilution factor of the cell suspension and can be calculated as follows:

F= (cell suspension 10 µl + trypan blue suspension x µl)/ cell suspension 10 µl

Transient transfection

To test whether the modified exon 79 fragment containing the in-frame FLAG-EGFP cassette was expressed properly, the fragment was subcloned into the mammalian expression vector pCMV-Tag3A and transfected into COS1 cells. The sequence of the Cox8a mitochondrial targeting signal was cloned 5' of the start codon of the cassette so that the exon 79*FLAG-EGFP fragment would be targeted into the mitochondria. Therefore, co-transfection with the mammalian expression vector pTag-RFP-mito was performed. This vector fluorescently labels the mitochondria with red fluorescence and co-localization with the EGFP signal would confirm the correct expression of the EGFP cassette. Transfection was carried out using the Lipofectamine® LTX reagent (LifeTechnologies) according to the manufacturer's instructions.

24 h after transfection cells were imaged for EGFP and RFP fluorescence.

Generation of Dmd EGFP reporter mice

The work comprising homologous recombination in ES cells, screening for correct insertion (using our predesigned Southern blot probes), blastocyst injection, as well as breeding of the F1-generation and screening for germline transmission was done commercially by the Polygene AG, Ruemlang, Switzerland.

ES cell electroporation, generation of chimeric mice and germline transmission

The targeting vector was linearized using SalI and electroporated into SV129Ola-derived ES cells. G418 (0.4 mg/ml) was used to select for stable transfection. and F2 generation were genotyped via PCR on DNA extracted from tail biopsies.

Genotyping of the mice

The DNA was extracted from the tail biopsies using the GenScript TissueDirect TM Multiplex PCR System (GenScript Corporation, Piscataway, NJ, USA) according to the manufacturer's instructions. Genomic DNA was obtained directly during lysis of the tissue without any further purification steps and used for PCR amplification with gene specific primers (Appendix 13.1, Table A1).

Sacrificing and sectioning of the animals

Donor animals were sacrificed by cervical dislocation. The dead animals were fixed in dorsal position on a cork-tray and disinfected with 70% ethanol. The fur was removed carefully and the following organs were prepared for cryopreservation and subsequent sectioning (section 6.5.1) or used for extraction of single fibers and satellite cells (section 6.5.2): Muscles:

tibialis anterior (TA), extensor digitorum longus (EDL), quadriceps (QC), gastrocnemius (GC), soleus (SOL) and diaphragm (DIA); as well as heart, stomach, gut, brain, and eyes.

Methods for analysis of the transgenic mice

Tissue and section preparation

Tibialis anterior (TA), extensor digitorum longus (EDL), quadriceps (QC), gastrocnemius (GC)

and soleus (SOL) muscles as well as the diaphragm (DIA), heart, ileum, duodenum, colon, stomach, brain, and eye were dissected from 8 to 12-weeks-old (adult) or 6-month-old male

Dmd EGFP mice and their age-and sex matched wildtype littermates. The tissue was mounted on Tissue-Tek® O.C.T. (Hartenstein, Germany), frozen in fluid nitrogen cooled isopentane and processed for cryostat sectioning. 8 µm cross sections were collected from the mid-belly of the muscles and either stored at -80°C or directly fixed and stained.

Isolation of EDL-muscle derived myofibers

EDL muscles were dissected from the hind limbs of 8-weeks-old Dmd EGFP mice and their wildtype littermates and digested in 0.2 % collagenase type I (Sigma-Aldrich, Germany) in DMEM as previously described [START_REF] Pasut | Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle [Internet[END_REF]. Muscles were transferred to DMEM-filled horse serum-rinsed Petri dishes using a heat-polished glass Pasteur pipette. Muscles were triturated until the fibers became separated, transferred to a fresh dish and incubated at 37 o C, 5% CO2. Intact fibers were transferred under the dissecting stereomicroscope into fresh DMEM-filled horse serum-rinsed dishes using a thinly bored Pasteur pipette to exclude any cell debris. The isolated myofibers were either fixed for immunofluorescence (IF) staining or kept in culture in for satellite cells differentiation.

Culture and differentiation of single fiber-derived satellite cells

The isolated myofibers including their attached satellite cells (SC) were cultured in DMEM supplemented with 20% fetal bovine serum (Sigma-Aldrich), 10% horse serum (HS, Sigma-Aldrich) and 1% chicken embryo extract (CEE, Ultrafiltrate, VWR International, Germany) in dishes that had been coated with 10% Matrigel® (BDBiosciences, Germany) in DMEM (Danoviz and Yablonka-Reuveni, 2012). After 2 days, satellite cells-derived myogenic progenitors started to migrate off the myofibers and formed myogenic colonies composed of proliferating myoblasts that differentiated into myotubes between days 6 and 8. This process was enhanced by changing the growth medium after 5 days to low horse serum (4% HS) medium. Differentiating myoblasts were observed daily under the microscope and fixed after 8 days for subsequent immunofluorescent staining.

Histological analysis and immunhistochemistry

8 μm cryosections from either adult or aged mice were stained with haematoxylin/eosin (H&E) at the institute of Neuropathology (Charité, Berlin).

8 µm cryosections from adult mice were analyzed via immunohistochemistry (IHC) and immunofluorescence (IF). Single fibers and differentiated myoblasts were analyzed via immunofluorescence.

Immunohistochemistry on cryosections was performed at the Institute of Neuropathology (Charité, Berlin) with primary antibodies and appropriate biotinylated secondary antibodies and diaminobenzidine visualization of the peroxidase reaction. In order to suppress unspecific background staining, we used the M.O.M. TM kit (Vector Laboratories, Burlingame, CA, USA) for all primary mouse antibodies. All dilutions of the primary antibodies used in this study are described in Appendix 13.5, Table A2-3.

Immunofluorescent staining was performed on single fibers, differentiated myoblasts and 8 µm muscle cryosections. Fibers and myoblasts were fixed in 4% PFA, washed, incubated for 1 h at room temperature in blocking solution (BS) containing 5% normal goat serum, 0.5% BSA and 0.2% Triton X-100), followed by overnight incubation with primary antibodies in BS. The cryosections were fixed in ice cold acetone, washed in PBS, blocked 30 min in 5% normal goat serum with followed by 1 h incubation with primary antibodies in 3% BSA in PBS. For staining with primary mouse antibodies cryosections were blocked additionally for 1 h with an immune globulin blocking reagent from the M.O.M. TM kit. After the primary antibody incubation, fibers, myotubes and cryosections were washed and incubated for 1 h with fluorescent dye coupled secondary antibodies (Alexa488 and 568, LifeTechnologies, CA, USA), washed three times in PBS and counter stained with DAPI.

Primary antibodies in this study were directed against the following proteins or proteindomains: laminin, β-spectrin, dystrophin, Dys2, Dys1, MANDYS19, H4, GFP, FLAG, GFAP, CD31, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, α-dystroglycan, nNOS, α-bungarotoxin, vinculin, α-actinin2 and utrophin. Images were recorded with an inverted fluorescent microscope (Leica DMI4000).

Western blot analysis

In order to analyze the expression of the dystrophin protein and potential isoforms in the muscles of transgenic mice we performed Western blotting. Proteins were extracted from the tissue of interest and separated by SDS-PAGE according to their molecular size. Proteins were then transferred onto a membrane by electro-blotting and incubated with an antibody specific for the protein of interest and visualized using a chemiluminescent reaction. Proteins were extracted from tibialis anterior (TA) muscle or brain of 8-weeks-old male Dmd EGFP mice and their wildtype littermates after homogenization in liquid nitrogen and sonification in lysis buffer (75 mM Tris, pH 6.8, 1% SDS) containing a proteinase inhibitor cocktail (Complete®, Roche-Diagnostics). The Western blot analysis was done at the Institute of Neuropathology (Charité, Berlin) and at the Université de Versailles St-Quentin (Montigny-le-Bretonneux, France). Briefly, 50 µg protein were loaded on a 3-8% tris-acetate gradient gel (Novex, LifeTechnologies), electrophoresed and wet-blotted onto a nitrocellulose membrane. The blots were probed with mouse antibodies against the dystrophin rod domain (Dys1) and the dystrophin C-terminus (Dys2) in 5% milk powder for 2 days at 4°C followed by incubation with the secondary goat-anti-mouse IgG peroxidase conjugated antibody. Bands were visualized by chemiluminescence. Subsequently, the blot was stripped and re-probed with a second mouse monoclonal anti-GFP antibody and rabbit polyclonal anti-α-actinin 2 antibody. For Western blots on brain tissue, protein extraction was done using a combination of following two buffers containing proteinase inhibitor cocktail: (i) 250 mM sucrose, 10mM Tris-HCl, pH 8.2 and (ii) 20% SDS, 20% glycerol, 10% beta-marcaptoethanol, 12,5% western blot running buffer in water. NuPage LDS sample buffer (Invitrogen) was used for the gel electrophoresis.

The blots were probed using the iBind Flex Western System (Invitrogen, LifeTechnologies) with a mouse antibody against the dystrophin rod domain (Dys1,) and a rabbit polyclonal antibody against the dystrophin C-terminus (clone H4, gift from Cyrille Vaillend) together with secondary fluorescent antibodies (Alexa Fluor 700 and 800 respectively). A second blot was probed with monoclonal anti-GFP antibody together with a secondary fluorescent antibody (Alexa Fluor 700). As loading control, a mouse monoclonal anti-vinculin antibody was used.

Bands were visualized using the Odyssey CLx system (Li-Cor). All antibodies are listed in the Appendix 13.5, Table A2-3.

RESULTS

Generation of Dmd EGFP reporter mice

The first part of my study was focused on the generation of a novel Dmd EGFP reporter mouse.

For this model, the gene encoding the Enhanced Green Fluorescent Protein (EGFP) had to be integrated into the Dmd locus after the last exon 79. This way, Dmd splice isoforms containing exon 79 would be automatically tagged with EGFP at their C-terminus. The dystrophin-EGFP fusion protein will facilitate the study of dystrophin expression in different tissues in vivo and ex vivo without the need for antibody staining.

The single steps leading to the generation of the Dmd EGFP reporter mice are summarized in Figure 5. In order to modify the murine dystrophin locus on the X-chromosome, a targeting vector was designed. The vector encodes a modified sequence of the last dystrophin exon (removal of the natural termination codon), an in-frame insertion of a FLAG-EGFP sequence, followed by a loxP flanked neomycin (neo) cassette in the 3'UTR. Flanking the targeting cassette, homologous Dmd stretches were inserted into the vector that will enable homologous recombination in embryonic stem cells (ES cells). Successfully targeted ES cells were used for blastocyst injection, generation of chimeric mice and germline transmission of the modified allele. The part of this work including the ES cell culture and generation of transgenic mice in the F1 generation was done by a commercial company (Polygene AG, Switzerland). Transgenic mice expressing the dystrophin-EGFP fusion protein were then crossed with ubiquitous Cre-deleter mice to remove the neo selection cassette. The targeting strategy for modification of the Dmd locus using the targeting vector is depicted in Figure 6.

All plasmid maps and primers used for cloning of the targeting vector and for genotyping the mice are attached in the Appendix 13.1 and 13.2. The cloning software Vector NTI® (Invitrogen, Germany) was used to visualize and in silico test the design strategy.

Subcloning of a genomic

Generation of the targeting vector

The results of the process of making the targeting vector are described in more detail in the next sections. The targeting cassette consists of three regions: (i) the region encoding the modified exon 79 together with the FLAG-EGFP tag, (ii) a neo resistance gene flanked by loxP sites and (ii) two homology arms enabling homologous recombination in ES cells.

Subcloning of a genomic Dmd fragment containing the coding sequence of Dmd exon 79

In order to modify the Dmd locus we used a bacterial artificial chromosome plasmid (BAC) DNA (Sv129 derived, bMQ389g17, Source Bioscience, Germany) covering the dystrophin genomic region comprising intron 78, exon 79, and part of the 3'UTR. We selected a 10.943 kb Dmd fragment around exon 79 for modification and subcloned it from the BAC into the pCR-Script-AMP vector to facilitate subsequent cloning steps. The subcloning was done by gap repair via recombineering (see section 6.2.5), a cloning method that uses homologous A targeting vector was constructed for insertion of a FLAG-EGFP coding sequence into the 3'UTR of the Dmd gene so that a dystrophin-EGFP fusion protein would be expressed. The last exon 79 was modified by exchanging the stop codon to a leucine (an asterisk indicates the modified exon 79, and a FLAG-EGFP sequence with a loxP-sites flanked neo cassette was inserted. The Dmd wildtype allele was targeted in ES cells after electroporation of the SalI linearized targeting vector and subsequent homologous recombination. Dashed lines represent homologous regions. BglII and BamHI restriction sites were artificially inserted into the transgene to facilitate identification of targeted ES cells by Southern blot analysis using either 5' and 3' hybridization probes (blue squares) located outside of the targeting vector. Positive ES cells were used for generation of transgenic mice. The neo cassette was removed by crossing the F1 generation mice with ubiquitous Cre-deleter mice (constructs are not drawn to scale). Ex: exon. recombination in bacteria without the need of restriction enzymes. Figure 7 shows the cloning strategy. Homology arms, no longer than 238 bp, flanking the selected Dmd region were amplified from the BAC clone (5' and 3' Homology Arms; HAs). We used 5'-tailed oligonucleotide primers for amplification that contained restriction enzyme recognition sequences enabling cloning of the arms into the pCR-Script-AMP vector. Thereafter, the pCR-Script+HAs vector was linearized using HindIII, a restriction enzyme cutting exactly between the two homology arms, leaving a gap. The linearized DNA was electroporated into recombination competent E. coli SW106 cells that carried already the BAC. Recombination between the HAs on the BAC and on the linearized pCR-Script+HAs vector led to gap repair of the plasmid and retrieval of the 10.943 kb Dmd fragment of interest. The ensuing vector was named Dmd exon 79-retrieval vector. The retrieval vector was then further modified in order to generate the targeting vector for homologous recombination in ES cells. Homology arms (HAs) were amplified from BAC DNA using primer pairs with the following restriction enzyme sites: Sall, NotI, and HindIII (5'HA) as well as HindIII and NotI (3'HA). HAs were cloned into the pCR-Script-AMP vector. In a second step BAC DNA was electroporated into E. coli SW106 cells. Subsequently, recombineering genes were induced and the HindIII linearized pCR-Script+HAs DNA was transformed into these cells. After successful recombination via gap-repair the 10.943 kb Dmd exon 79 fragment was cloned into the pCR-Script+HAs vector generating the Dmd exon 79-retrieval vector. Positive clones were selected by their ampicillin (Amp r ) resistance. Ex: exon, HA: homology arms.

Generation of a mini targeting cassette

For the modification of the retrieval vector by inserting the FLAG-EGFP coding sequence and the loxP flanked neo cassette and thus generating the targeting vector, a mini targeting cassette was cloned. As a recipient of the mini targeting cassette, we used the PL452 vector that already contained a loxP flanked neo resistance gene, which is transcriptionally controlled by a hybrid PGK/Em7 promoter. PGK drives neo expression in mammalian cells and Em7 permits the expression of neo in bacterial cells. A modified Dmd exon 79-FLAG-EGFP sequence was inserted upstream of the loxP-neo-loxP cassette. Furthermore, mini homology arms were designed and cloned into the vector to enable recombination. A mini targeting cassette was designed in such a way that the two homology arms (5' and 3' mini homology arms, 200 bp each) span DNA regions next to each other on the genomic Dmd exon 79 fragment, and the 5' mini arm includes exon 79. The 3' mini arm starts downstream of the stop codon sequence in exon 79.

Both homology arms were amplified from the BAC DNA and cloned into the PL452 vector using 5'tailed oligonucleotide primers as already described and shown in Figure 7. During the amplification of the 5' mini arm modifications were introduced via the reverse 3' primer: (i) the primer binds to the exon 79 sequence, however, introduces a single nucleotide mismatch changing the natural stop codon of exon 79 into a leucine coding sequence (TAGStop>TTGLeu), (ii) this long primer further contained the FLAG sequence fused to the exon 79 sequence and (iii) a unique BglII site for later Southern blot analysis as well. The 5' mini homology arm thus still contains the Dmd homology region of 200 bp but with a slight modification of the exon 79 stop coding sequence and a FLAG coding sequence. The arm was inserted upstream of the neo cassette in into PL452 vector. The 3' mini homology arm, which contains a region from the 3'UTR was cloned downstream of the loxP flanked cassette. The vector PL452+mini HAs was generated (Figure 8).

In the next step, an EGFP sequence was cloned into the vector downstream of the FLAG sequence. EGFP was amplified from the plasmid pEGFP-N3-L221K again using 5'-tailed oligonucleotide primers. This vector encodes a monomeric version of EGFP in which a hydrophobic residue is exchanged for a positively charged residue (L221K). EGFP was digested with EcoRI and cloned into the PL45+mini HAs (Figure 8). The new vector contained the mini targeting cassette consisting of the modified Dmd exon 79-FLAG-EGFP sequence (inserted in-frame) and the loxP flanked neo cassette. The homology arms flank this cassette and can be used for recombineering (Figure 8 ).

Generation of the targeting vector

In order to generate the final targeting vector, the mini targeting cassette was digested from the PL452 vector and electroporated together with the Dmd exon 79-retrieval vector into recombineering competent E. coli SW106 cells. Successful recombination of mini homology arms resulted in the modification of the Dmd exon 79 retrieval vector by inserting the modified exon 79 sequence followed by FLAG-EGFP-loxP-neo-loxP cassette between the two mini homology arms (Figure 9). Thus, the targeting vector was generated and could be used In the first step (1.) 5' and 3' mini homology arms were amplified from BAC DNA using 5'-tailed oligonucleotide primers: for the 5' mini arm spanning exon 79, KpnI and EcoRI-BglII-FLAG-modified exon 79* containing primers were designed. The 3' mini arm contained the BamHI and SacII recognition sequences. After amplification, the 200 bp mini arms together with the PL452 vector were digested and the fragments were inserted: the 5' mini arm with exon 79*-FLAG upstream of the loxP-neo cassette, and the 3' mini arm downstream of the loxP flanked neo cassette. In a second step (2.) an EGFP sequence was amplified with EcoRI flanked primers from pEGFP-N3-L221K and cloned into PL452+mini HAs downstream of the FLAG sequence generating the PL452+mini targeting cassette. A unique BglII site was included for later Southern blot analysis. Asterisk (*) indicates the modified exon 79. Ex: exon, HAs homology arms, Amp r : ampiciline resistance, NEO r : neomycine resistance.

for electroporation into embryonic stem cells and modification of the murine dystrophin locus. The plasmid maps of all vectors used in the cloning strategy are depicted in Appendix 13.2, Figure A1.

In vitro testing of the targeting vector

To confirm proper function of the targeting vector I performed two in vitro functional tests.

Modified Dmd exon79-FLAG-EGFP cassette

The modified exon 79-FLAG-EGFP coding sequence was tested for in-frame insertion and proper EGFP-expression in mammalian cells. For that, the cassette was amplified from the mini targeting vector using specific tailed primers and was subcloned into a mammalian expression vector (pCMV-Tag3A). A Cox8a mitochondrial targeting signal sequence was inserted upstream of the Dmd cassette so that the EGFP fluorescence signal should be The mini targeting cassette was digested from the PL452 vector using KpnI and SacII restriction enzymes and electroporated together with the Dmd exon 79-retrieval vector into E. coli SW106 cells, in which the recombineering genes had been induced. Homologous recombination resulted in the insertion of the mini targeting cassette into the retrieval plasmid, thereby modifying the Dmd fragment. The targeting vector can be linearized with SalI. Ex: exon, HAs homology arms, Amp r : ampiciline resistance, NEO r : neomycine resistance.

localized only in the mitochondria after transfection of COS cells. A plasmid expressing red fluorescent protein (RFP) only in the mitochondria (Mito-RFP, Evrogen) was co-transfected and was used as localization control. Figure 10 shows the expression of the two plasmids and their co-localization in the mitochondria confirming proper expression of the targeting cassette.

Excision of the neo cassette

The loxP flanked neo cassette in the targeting vector allows kanamycin selection after transfection into ES cells. However, after successful targeting of the genomic allele, it has to be excised in order to prevent unpredicted interference at the Dmd locus. This has to be done by breeding the F1-mice with ubiquitously expressing Cre-deleter mice. This leads to the excision of the loxP site-flanked neo cassette in the germ-cells of the succeeding F2generation. Hence, the recognition sequence and its functionality had to be confirmed in vitro as well. The modified Dmd exon 79 targeting vector was electroporated into E. coli SW106 bacteria. In these cells, the expression of the Cre-recombinase was induced by growing them on arabinose containing medium. Clones were analyzed for the excision of the neo cassette using colony PCR (Figure 11A) with specific primers. The amplified fragments were visualized on agarose gel. Figure 11B (lanes 7-9) shows three positive clones in which a 500 bp fragment was amplified only if recombination had taken place. A second PCR using a primer that binds in the neo cassette shows no bands in these three clones, thus confirming the excision.

Electroporation of ES cells and selection of positive ES cell clones

The targeting vector was linearized using SalI and electroporated into 129-derived ES cells.

After G418 selection, positive clones were picked and analyzed via Southern blot analysis. The screening strategy and the probes for the Southern blot were provided to the company by me.

Screening for positive ES cell clones via southern blot analysis

For the Southern blot analysis, I generated 5' and 3' oligonucleotide probes that were located outside of the 10.943 kb homologous regions (Figure 12A, for plasmid maps of the subcloned probes, see Appendix 13.2, Figure A1). For the analysis, unique restriction enzymes recognition sites were used that had previously been artificially inserted into the targeting vector. A 5' DNA probe detects a 15.9 kb fragment in BglII digested genomic DNA if the wildtype allele is present, whereas in the targeted allele the probe binds to a 6.2 kb fragment (Figure 12B). Ten positive ES clones with a correctly targeted allele were identified via Southern blot with 5' probe. DNA from 6 of these clones was further tested using the 3' probe after digestion with BglII or with BamHI in order to confirm the successful recombination at the 3' side of the construct. The ES cells used for homologous recombination were from male donors and since Dmd is located on the X-chromosome, we expected to see only one band.

Nevertheless, the presence of a second wildtype band was visible in some of the positive ES clones suggesting either incomplete digestion by the restriction endonucleases or a contamination with wildtype DNA, possibly due to microheterogeneity. (A) 5' and 3' DNA probes (blue squares) complementary to a genomic region outside of the targeting cassette were generated. In the wildtype allele the 5' and 3' probes detects a 15.9 kb band after digestion with BglII, whereas in the targeted allele a 6.2 or 12.4 kb band can be detected, respectively. Using BamHI for DNA digestion the 3' probe hybridized to an 18.5 kb fragment in the wildtype and to a 14.3 kb fragment in the targeted cells. (B) DNA from ES cell clones was digested with BglII and hybridized with the 5' probe. Ten positive clones with the recombinant allele are numbered and marked with red (left blot) and two wildtype clones were numbered and marked in black. The DNA from positive clone 2, 3, 4, 6, 7 and 11 was further hybridized with the 3' probe after BglII and BamHI digestion (right blots). As control, wildtype clone 1 and 9 (black) were tested. Clone 6 marked in red with an asterisk was the one that later led to the successful generation of chimeric mice and germ line transmission. Wildtype DNA was used as control. WT: wildtype, M: marker, kb: kilobase-pair, Ex: exon.

Generation of chimeric mice via blastocyst injection of positive ES cell clones

Positive ES cell clones were injected into C57BL/6N blastocysts. Surviving blastocysts were transferred into CD1 foster mice, which gave birth to mice with different percentages of coat color chimerism. Good chimeras (>80%) were picked and mated to C57Bl/6N mice. After several rounds using different positive ES cell clones for blastocyst injection, clone 6 gave rise to good chimeras that were mated to C57Bl/6N mice and germline transmission of the targeted allele was achieved (Figure 13).

Confirmation of germline transmission via genotyping and Southern blot analysis

Agouti offspring from the mating of chimeric with C57BL/6N mice was screened for the presence of the neo cassette. Specific primers (Appendix 13.1, Table A1) were used for the screening PCR. A positive PCR reaction obtaining a 542 bp band was taken as evidence for germline transmission of the targeted dystrophin allele. Three animals of the F1 generation and offspring of chimera 14 were analyzed. Agarose gel electrophoresis of the PCR amplified fragments is shown in Figure 14A. In animals 1018 and 1019 the neo fragment was amplified, suggesting germline transmission, whereas animal 1020 did not show any band corresponding to the wildtype allele. To further confirm the PCR results, DNA was prepared from tail biopsies, digested with the enzyme BglII, Southern blotted, and labeled with the 5' probe as in section 7.1.5.1. In confirmation of the PCR screening, the targeted fragment of 6.2 kb could only be detected in the animals 1018 and 1019, whereas animal 1020 showed the wildtype 15.9 kb band (Figure 14B). As expected all positive mice were females as the targeted allele is located on the X-chromosome.

Excision of the neo cassette by crossing the Dmd EGFP-neo mice with Cre-deleter mice and genotyping of the F2 generation

After confirmation of the presence of the targeted allele in the F1 generation, the mice were sanitized and entered into a breeding program at the SPF facility at the Forschungseinrichtungen für Experimentelle Medizin (FEM) of the Charité via embryo transfer.

In order to remove the neo-cassette, animals from the F2 generation were mated with Credeleter mice, which ubiquitously expressed the Cre-recombinase. This enzyme excises the neo-sequence between the two loxP sites in the whole body, including the germ cells. Mice from the F3 generation were genotyped via tail biopsy PCR (Figure 15). The genotype of the transgenic Dmd EGFP mice was verified by a three primer PCR that allows the distinction between wildtype, female heterozygous and homozygous as well as male hemizygous animals. Figure 15B shows an agarose gel electrophoresis of PCR products from different animals (1-8). Amplification of the wildtype allele yields a 202 bp band (lane 1-3) while amplification of the Dmd EGFP allele resulted in a 304 bp band (lane 4, 5, 6 and 8). In lane 6 both fragments were detected suggesting that the animal is a heterozygous female.

Characterization of Dmd EGFP reporter mice

After verification of the successful targeting and modification of the dystrophin locus the Dmd EGFP reporter mice were characterized. Transgenic mice were viable and did not show any obvious phenotypic defects. We investigated the expression and localization of the dystrophin-EGFP fusion protein throughout different tissues exploiting the natural EGFP fluorescence. The C-terminus of dystrophin is an important functional domain and it is expressed in the most dystrophin isoforms as well as in revertant dystrophin. Introducing a FLAG-EGFP tag of 27.9 kDa at its C-terminus might have an impact on the muscle physiology and potential dystrophic changes had to be ruled out. Therefore, I analyzed muscle histology as well as the location and expression of dystrophin interaction partners from the DAPC. (A) A three primer PCR was used for genotyping. Primer S4228 binds to intron 78, primer S4269 binds to the EGFP sequence and primer S4229 to the 3'UTR. A 202 bp band is only detectable in samples from wildtype animals (S4228 and S4229), amplification using primer S4269 and S4229 will generate a 350 bp band only from the recombinant allele, after removal of the neo cassette. (B) Agarose gel electrophoresis of the genotyping PCR. Animals 1-3 had the wildtype allele and were either male (-/y) or female (-/-). In Dmd EGFP homozygous male (+/y) and female (+/+) mice the 350 bp band is detected (lanes 4,5,[START_REF] Watkins | Immunoelectron microscopic localization of dystrophin in myofibres[END_REF]8). In heterozygous females (+/-) both amplicons were detected (lane 6). Ex: exon, bp: base pair, M: Marker, WT: wildtype. satellite cells. For the experiments we used 8-12 weeks old male Dmd EGFP reporter mice and their wildtype littermates. Histological changes that would only come with age were screened in animals that were 6 months or older.

Furthermore, I investigated the EGFP expression in isolated myofibers and in differentiating

Expression of the tagged dystrophin-EGFP protein

The expression of native EGFP was observed in all skeletal muscles of the Dmd EGFP reporter mice at sarcolemmal level. Figure 16 We confirmed the correct expression of the EGFP-tagged dystrophin by Western blot analysis using dystrophin antibodies against the rod-and C-terminal domains (Dys1 and Dys2 respectively) (Figure 18). The bands from wildtype dystrophin and from dystrophin-EGFP had the same expression intensities detected by the rod-domain specific antibody, and the band intensity of the Dmd EGFP samples was a bit lower when we used the Dys2 antibody. The molecular sizes of the proteins detected in both samples were comparable, since differences in molecular weight of 27.9 kDa between full-length dystrophin and dystrophin-EGFP would not be detectable on the Western blot at molecular weights (MW) above 400 kDa. An EGFP band in the dystrophin molecular size range was only detected in the Dmd EGFP muscle sample.

Expression and localization of dystrophin-EGFP in different tissues

In the next experiments we analyzed the expression and localization of dystrophin-EGFP in different tissues to confirm the tagging of different dystrophin isoforms.

Dystrophin-EGFP in skeletal muscle

We confirmed the correct localization of native EGFP fluorescence at the sarcolemma in TA, gastrocnemius (GAS) and soleus (SOL) muscles. Co-immunostaining with the dystrophin antibodies MANDYS19 (against the rod domain) and Dys2 (against the C-terminal domain) as well as with the cytoskeletal anti-β-spectrin antibody, showed an exact overlay of the fluorescent signals (Figure 19A-C). Importantly, the comparison between signals for EGFP and laminin, which is an extracellular matrix protein, reveals -as expected -a distinct staining pattern with the EGFP signal being present more towards the interior of the fiber (Figure 19D).

The anti-β-spectrin and anti-laminin antibodies stained also the capillaries that did not show any EGFP signal. Other skeletal muscles showed a similar EGFP expression pattern (data not shown).

Dystrophin-EGFP in cardiac tissue

We confirmed the correct dystrophin-EGFP expression and localization in the heart by verifying co-localization of the EGFP-signal with the signals generated by anti-dystrophin immunofluorescent staining (Figure 20A). Furthermore, EGFP expression was detected in blood vessels (arteries or veins) of the cardiac muscle that stained positive with anti-CD31 antibody directed against platelet endothelial cell adhesion molecule (Figure 20B). This staining pattern suggests the expression of dystrophin-EGFP in the smooth muscles of the vessel walls.

Dystrophin-EGFP expression in smooth muscle

We also analyzed the EGFP expression of dystrophin in the smooth muscles of reporter mice.

Cross sections of ileum, duodenum, stomach and colon revealed a positive fluorescent signal in the circular and longitudinal smooth muscle cell layers (Figure 21A). Additionally, colon and

Dystrophin-EGFP expression in non-muscle tissue

In order to investigate whether the different dystrophin isoforms were tagged in non-muscle tissues as well, we analyzed the EGFP expression in brain and retina of Dmd EGFP mice. We performed a Western blot analysis on whole brain lysates from reporter mice and wildtype littermates (Figure 22). The full-length Dp427 isoform was detected at a very low level in wildtype samples using the C-terminal anti-dystrophin antibody H4. In the same sample a strong band corresponding to Dp71 was detected by the H4 antibody. In the brain lysates of reporter mice the Dp427 isoform was not detected using either H4 or anti-GFP antibodies. folding, its subcellular localization, and protein function, thereby possibly provoking a muscular dystrophy similar to that seen in the mdx mouse model of Duchenne muscular dystrophy.

Muscle morphology

We performed H&E staining in adult and aged transgenic mice and their wildtype littermates. This investigation revealed normal skeletal muscle morphology in adult and aged Dmd EGFP mice and the entire absence of dystrophic changes such as increased myofiber size variability, fibrosis and centrally located nuclei (Figure 25, Appendix 13.3, Figure A2). Thus, histological analyses confirmed that EGFP tagging of the dystrophin did not cause any overt muscle pathology, neither in young mice nor with advancing age.

Expression of DAPC members

In a next step, we investigated whether the introduction of the C-terminal EGFP tag would influence the correct assembly of the DAPC. We hence investigated the expression of the following DAPC members in different skeletal muscles: dystrophin, α-dystroglycan, α-, β-, γsarcoglycan, nNOS, as well as utrophin (Figure 26, Appendix 13.4, Figure A3). Figure 26 depicts normal sarcolemmal expression of γ-sarcoglycan, α-dystroglycan and nNOS in TA muscle sections. However, expression levels differed between individual sections because we used DAB-mediated antibody labeling to circumvent autofluorescence. Utrophin upregulation can be observed in DMD patients as well as in mdx mice, and it is used as an unspecific marker for muscle dystrophy. However, we did not find any upregulation of utrophin in the Dmd EGFP TA muscle, further confirming that the dystrophin-EGFP fusion protein did have a negative impact on muscle integrity (Figure 26). The same expression pattern was observed in the GAS, QUAD, SOL and EDL muscles (Appendix 13.4, Figure A3 ).

Ex vivo and in vitro expression of dystrophin-EGFP in single fibers and differentiating satellite cell-derived myoblasts

Another interesting question would be whether the presence of dystrophin-EGFP would allow the live-imaging of dystrophin expression in cultures of isolated myofibers. Indeed, isolated myofibers from Dmd EGFP were strongly EGFP fluorescent standing in contrast to the entire absence of an EGFP signal from wildtype fibers (Figure 27A). In addition, strong dystrophin-EGFP expression was detected at the neuromuscular junction of single fibers that have been co-stained with fluorescently labeled α-bungarotoxin (BTX) that marks acetylcholine receptors (AChR). Interestingly, the EGFP expression at the NMJ of fixed fibers was stronger then the signal observed at this structure after BTX staining procedure (Figure 27B-C).

Further, we tested whether dystrophin-EGFP would be increasingly expressed in cultures of differentiating satellite cell-derived myoblasts. For this, we cultured isolated myofibers together with their attached satellite cells. As previously reported [START_REF] Pasut | Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle [Internet[END_REF], satellite cells detached from their fiber, proliferated and formed myotubes after 8 days in differentiation medium. However, native EGFP-fluorescence was too weak to be detected in single myotubes. Remarkably, co-immunocytochemistry using both anti-GFP and antidystrophin antibodies demonstrated an extremely intense GFP signal, which co-localized with dystrophin (Figure 28). Of note, the anti-dystrophin signal in the myotubes was very weak, a known disadvantage of studying dystrophin in vitro. Thus, Dmd EGFP derived myogenic precursor cells are capable to produce the dystrophin-EGFP in vitro and the signal is superior to the classic anti-dystrophin antibody-mediated dystrophin signal.

Generation of Dmd EGFP reporter mice

The development of genetically modified animal models has contributed vastly to the advancement of biomedical and human disease research. Genetic and physiological similarities between humans and mice have placed the mouse as an important mammalian model for numerous studies in health and disease. These models have extended our understanding of basic biological processes, disease states and have led to the development of therapeutic strategies.

In the late 70ies and beginning of the 80ies, different groups developed strategies for the introduction of exogenous DNA into the murine genome conferring Mendelian inheritable traits [START_REF] Costantini | Introduction of a rabbit beta-globin gene into the mouse germ line[END_REF][START_REF] Gordon | Integration and stable germ line transmission of genes injected into mouse pronuclei[END_REF][START_REF] Harbers | Microinjection of cloned retroviral genomes into mouse zygotes: integration and expression in the animal[END_REF][START_REF] Palmiter | Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes[END_REF][START_REF] Stewart | Successive generations of mice produced from an established culture line of euploid teratocarcinoma cells[END_REF][START_REF] Tremblay | A Case for Immunosuppression for Myoblast Transplantation in Duchenne Muscular Dystrophy[END_REF][START_REF] Wagner | The human beta-globin gene and a functional viral thymidine kinase gene in developing mice[END_REF]. The method used to achieve this result was the introduction of specific cloned sequences into the pronuclei of zygote stage embryos via microinjection. The manipulated embryos were then transferred into pseudo-pregnant recipient females, went through normal embryonic development and were born as viable offspring. The huge potential of this novel technology led to the rapid development in this research field, improving and optimizing the method.

Transgenic lines have been developed expressing specific genes both in spatially and temporally defined manner. Abrogation and overexpression of disease causing genes have been investigated extensively in mouse models with engineered gene specific mutations.

Further progress was achieved with the identification and development of embryonic stem (ES) cell cultures that were established in parallel by independent researchers [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF][START_REF] Smithies | Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination[END_REF][START_REF] Thomas | High frequency targeting of genes to specific sites in the mammalian genome[END_REF]. With the help of molecular genetic manipulation of ES cells, endogenous genes can be deleted and/or modified, a strategy called "gene targeting" that enables the generation of knockin/knockout mice. Injection of these ES cells into blastocysts and subsequent re-implantation into the murine uterus may then lead to offspring with germline transmission of the transgene to subsequent generations of the transgenic animals. We used this strategy to modify the murine dystrophin gene through inframe insertion of a fluorescent tag behind its C-terminal coding sequence to trace and visualize dystrophin in vivo without the need for immunohistological staining.

A large majority of the studies on DMD as well as most of our understanding about the function of dystrophin in normal and diseased state have been gained through mouse models.

The first and still most widely used mouse model for DMD is the natural mutant mdx mouse.

Additionally, various other dystrophic models have been developed on different genetic backgrounds [START_REF] Krivov | A new genetic variant of mdx mice: study of the phenotype[END_REF][START_REF] Schmidt | DNA Damage, Somatic Aneuploidy, and Malignant Sarcoma Susceptibility in Muscular Dystrophies [Internet[END_REF][START_REF] Wasala | The FVB Background Does Not Dramatically Alter the Dystrophic Phenotype of Mdx Mice[END_REF] and bearing different Dmd mutations [START_REF] Araki | Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy[END_REF][START_REF] Chapman | Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice[END_REF]. Most of the studies on the structure and function of dystrophin have been conducted on transgenic reporter mice using cDNA expression vectors. These models offer many advantages and have provided important information for gene therapeutic approaches, however, they have limitations and disadvantages. Usually tissue specific promoters are used to restrain the expression of the transgene. Despite this precaution, the transgene expression might still not exactly mirror the expression from the natural locus. Therefore, the protein expression patterns might considerably deviate from the natural situation. Moreover, these methods usually bypass endogenous control mechanisms of gene expression such as cell cycle regulation of transcription, microRNA regulation of translation, position effect or alternative splicing [START_REF] Ciotta | Recombineering BAC transgenes for protein tagging[END_REF]. To circumvent these limitations and the toxic effects by transfection and overexpression, we generated a model by which the fluorescent labeled protein is expressed throughout muscle and non-muscle tissue by the natural endogenous promoters. Up to date, no dystrophin reporter mouse model exists, in which the endogenous natural protein was tagged and could be traced and analyzed. For other animal models, only one study that has been published this year described a zebrafish gene trap line, in which a fluorescent citrine gene sequence is spliced in-frame into the rod-domain of zebrafish dmd [START_REF] Ruf-Zamojski | Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line[END_REF]. Furthermore, only few models exist in which transgenic dystrophin expression can be followed by means of a fluorescent tag that would greatly facilitate such investigations [START_REF] Bajanca | In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis[END_REF][START_REF] Li | A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy[END_REF]. The huge size of the dystrophin gene spanning 2.4 Mbp and encoding 79 exons constitutes a challenge and may explain why up to date so few reporter models for in vivo real time monitoring of dystrophin expression exist. Here we describe the generation and characterization of a Dmd EGFP reporter mouse, in which the fluorescent EGFP protein was fused to the C-terminus of endogenous dystrophin, hence targeting all major dystrophin isoforms that are expressed in different muscle and non-muscle tissues.

C-terminal tagging of the murine endogenous dystrophin with a fluorescent protein

For the generation of the Dmd EGFP reporter mouse, we used the method of gene targeting via The Dmd gene is a huge gene with a high degree of complexity in its regulation. At least 8 different promoters regulate the expression of tissue specific isoforms, which are beyond that also developmentally regulated. Furthermore, alternative splicing from each promoter contributes to diversity of dystrophin expression. The last Dmd exon 79 is present in all major dystrophin isoforms. In order to create a more "general" reporter model, the best position for insertion of the fluorescent tag sequence was deemed 3' of the Dmd gene in its 3'UTR.

Expression of the fluorescent tag at the C-terminus of the protein will enable the labeling of most of the dystrophin isoforms. Furthermore, in revertant dystrophin exon 79 is rarely spliced out. Further issues have to be addressed and concern the location of such a fluorescent tag with respect to the functional domains of the tagged protein. In dystrophin, the C-terminal domain harbors the most important functional domains of the protein and the insertion of the tag might influence protein structure and function. However, in vitro experiments using reverse transfection microarrays showed that protein tagging with GFP at its C-terminus in general is superior to tagging of the N-terminus [START_REF] Palmer | Investigation into the use of C-and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays[END_REF] with respect to correct protein localization, suggesting that the main functional characteristics might be maintained.

The second important question in the development of the targeting strategy was the choice of the fluorescence tag. In the ideal case, the signal from the tag will replicate the signal from a high affinity antibody or would be even more accurate with regard to its subcellular location because unspecific staining could be ruled out. A further advantage of such a fluorescent tag would be its detectability without the need for immunohistological staining that would open the possibility for live-cell imaging and investigation of dynamic processes.

A milestone in the transgenetic research was achieved by the cloning of the gene that encodes the green fluorescent protein (GFP) [START_REF] Prasher | Primary structure of the Aequorea victoria green-fluorescent protein[END_REF] 30 years after its discovery in the luminous hydromedusan, the jellyfish Aequorea victoria [START_REF] Shimomura | Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea[END_REF]. Since then, GFP was used as a fluorescent in vivo marker in bacteria, C. elegans, mice and in other living organisms to study and visualize cells and their behavior [START_REF] Chalfie | Green fluorescent protein as a marker for gene expression[END_REF][START_REF] Okabe | Green mice' as a source of ubiquitous green cells[END_REF]. Many modifications and optimizations have led to the development of modified versions of the Aequorea-based GFP with different emission spectra or chemical properties.

The enhanced GFP (EGFP) is among the brightest and most photostable fluorescent proteins.

For the tagging of the murine dystrophin gene, we hence opted for EGFP. The only drawbacks of EGFP as a fusion tag are its slight sensitivity to pH and a weak tendency to dimerize, the latter being a characteristic common to all GFPs.

Skeletal muscle dystrophin is a cytoplasmic protein located in the vicinity of the sarcolemma where it is part of a huge multimeric complex. Dystrophin interacts with its C-terminus with integral membrane proteins of the sarcolemma as well as with cytoplasmic proteins thus lending mechanical stability and serving as a scaffold for signaling molecules. Dimerization of EGFP at the C-terminus of dystrophin could thus be a major confounding factor and might have a negative impact on the function of the fusion protein. Furthermore, dimerization could lead to incorrect assumptions and measurements when applying advanced quantitative fluorescence techniques such as fluorescence resonance energy transfer (FRET). Therefore, we used a modified EGFP version in which dimerization was prevented. It has been shown that all Aequorea derived GFPs and mutants of any color contain a hydrophobic patch of Ala206, Leu221, and Phe223 responsible for dimerization of the beta-barrel. To circumvent an unwanted dimerization, we used a mutated EGFP (mEGFP) version in which the hydrophobic Leu221 was exchanges by the positively charged residue lysine. No significant alteration of the spectral properties have been reported for the monomeric mGFPs [START_REF] Zacharias | Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells[END_REF].

In order to use a fluorescent tag that will be useful for tracing a protein of interest, the concentrations of the fluorescent protein that are required for visualization over background fluorescence in cells have to be considered. In order to achieve a twofold increase in fluorescence over background, fluorescence EGFP must be expressed at minimum at 200 nM [START_REF] Patterson | Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy[END_REF][START_REF] Snapp | Design and Use of Fluorescent Fusion Proteins in Cell Biology[END_REF] which is the case for skeletal and heart muscles.

Recombineering: a rapid method for the generation of a targeting vector

To generate Dmd EGFP reporter mice, we tagged the murine Dmd gene with FLAG-EGFP sequence exactly adjacent to the C-terminal Dmd coding sequence behind exon 79. The process of generating the Dmd EGFP reporter mice involved different successive steps that are summarized in Figure 5. In the first part, a targeting vector was designed. We also inserted a FLAG sequence before the fluorescent tag sequence so that a broader range of experiments could be conducted with the reporter mice. FLAG is a short sequence of an antigenic peptide for which good antibodies exist and that can be used for protein tagging. This facilitates localization studies by immunohistochemistry and their purification as well as identification of binding partners [START_REF] Hopp | A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification[END_REF].

Applying the method of gene targeting, we used a cassette containing a modified sequence of Dmd exon 79 (with the stop codon removed) and FLAG-EGFP sequence followed by loxP flanked neo cassette for targeting the wildtype dystrophin allele in murine ES cells via homologous recombination. The targeted allele was then transferred to mice progeny and through crossing them with ubiquitous Cre-deleter mice the loxP flanked neo cassette is excised creating the desired "pure" knock-in allele (Figure 6).

For the introduction of the modified sequence into the genome, the natural process of homologous recombination was exploited. It is a type of genetic recombination, a universal biological mechanism, present in all domains of life. Nucleotides are exchanged between identical or similar DNA molecules. Such mechanism is used to repair double strand breaks, to create newly arranged DNA sequences during meiosis thereby contributing to genetic variation in the offspring and in horizontal gene transfer in bacteria and viruses [START_REF] Bibliography Alberts | General Recombination[END_REF]. For successful recombination and identification of positive clones as well as for confirmation of the modified allele's presence in ES cells and mice, different elements have to be constructed and engineered into the targeting cassette by cloning. Cloning is a time consuming process and different methods exist to speed up this process. We applied the method of "recombineering", which greatly facilitates the cloning procedure since it is independent of the presence of natural restriction endonuclease recognition sites. Knock-in vectors can thus be rapidly constructed from BAC clones via recombineering followed by gaprepair [START_REF] Murphy | Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in Escherichia coli[END_REF][START_REF] Zhang | A new logic for DNA engineering using recombination in Escherichia coli[END_REF]. We used a commercial BAC clone spanning the genomic DNA sequence of the murine Dmd containing exon 79. BAC clones are large (>100 kb) and thus not very suitable for modification and cloning steps. Hence we subcloned a smaller fragment of 10.943 kb DNA surrounding exon 79 into a pCR-Script vector via recombineering [START_REF] Liu | A highly efficient recombineering-based method for generating conditional knockout mutations[END_REF]. Modification of Dmd exon 79 was facilitated on this retrieval vector, which contains long enough stretches of homology upstream and downstream of exon 79 to ensure recombination in ES cells and targeting of the dystrophin allele. The homology arms on the targeting construct were around 3 kb for the 5' arm and 8 kb for the 3' arm. Arms of this size have proven to be sufficient to achieve good recombination with a high frequency [START_REF] Hasty | The length of homology required for gene targeting in embryonic stem cells[END_REF]. In order to fuse the FLAG-EGFP tag to the C-terminus of dystrophin, the sequence of the tag was inserted directly behind the last Dmd exon 79. The nucleotide sequence of the stop codon was modified so that the exogenous DNA could be read through and co-expressed as a fusion protein. For both recombineering steps (i) the generation of the retrieval vector and (ii) the modification of the retrieval vector, short homology arms had to be used to enable recombination. Although homologies of 50 flanking base pairs are more than sufficient for recombination, we used longer homology arms of 150-200 base pairs to further improve the targeting efficiency, which substantially decreased the effort required for subsequent screening of correct recombinants [START_REF] Murphy | Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in Escherichia coli[END_REF][START_REF] Sharan | Recombineering: a homologous recombination-based method of genetic engineering[END_REF]. To obtain these homology arms, we amplified each homology stretch by PCR and directionally cloned it into the plasmid by "classic" cloning using restriction endonucleases and DNA-ligase. The targeting vector now contained, besides the long arms of homology, the modified exon 79 followed by the FLAG-EGFP sequence and further downstream a loxP flanked neo sequence controlled by two promoters, which drive the expression of the selectable marker in prokaryotic as well as in eukaryotic cells. This neo cassette would later be used for screening of recombinant ES clones using G418. The selectable marker would be no longer necessary after ES cell selection and was excised in the F1 generation of the transgenic mice via a cross with Cre-deleter animals. This was done to make sure that the neo cassette would not exert any unwanted positional effect on the Dmd gene or on any other downstream genes. In the targeting vector the loxP flanked neo cassette was tested for its functionality shown in Figure 11. Furthermore, we engineered unique restriction enzyme sites into the transgene. The SalI restriction enzyme site was not present in the targeting cassette and would be located outside of the targeting region of the vector so that the vector could be linearized and electroporated in ES cells. In the targeting cassette a BglII and BamHI site were introduced that would assure the distinction between wildtype and modified allele in genotyping of the ES-cells via Southern blot. Those endonuclease restriction sites had to present only once in the targeting vector but also in the genome close to but outside of the recombination cassette (Figure 12).

Another important issue that we had to keep in mind while constructing the recombination cassette, was to prevent too harsh an alteration of the Dmd 3'UTR through insertion of the FLAG-EGFP-loxP-neo-loxP sequence. Important regulatory sequences are located in this 3'UTR region, which are highly conserved in the dystrophin gene [START_REF] Greener | The 3'-untranslated region of the dystrophin gene -conservation and consequences of loss[END_REF][START_REF] Larsen | Conserved regions of the DMD 3' UTR regulate translation and mRNA abundance in cultured myotubes[END_REF][START_REF] Lemaire | The chicken dystrophin cDNA: striking conservation of the C-terminal coding and 3' untranslated regions between man and chicken[END_REF].

Modifying the endogenous dystrophin gene in ES cells and generation of transgenic mice

We used the constructed targeting vector to modify the dystrophin locus in murine 129Sv ES cells. ES cell lines for genetic manipulations are derived from the inner cell mass of 3.5-dayold mouse embryo and the first line successfully grown in vitro was established in the early 80ies [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF]. The most successful ES cell lines were generated from the 129/Sv mouse strain, which is still the most widely used one [START_REF] Schoonjans | Improved Generation of Germline-Competent Embryonic Stem Cell Lines from Inbred Mouse Strains[END_REF]. ES cells are pluripotent and characterized by their capacity to self-renew. An additional characteristic of the ES cells is their ability to integrate into a developing mouse embryo when injected into a blastocyst [START_REF] Bradley | Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[END_REF]. They contribute to the developing embryo and produce the chimerism in the so generated mice. The contribution of the ES cells to the germline cells of the chimeric mice is dependent on the truly totipotent state of the ES cells in the culture, an important condition in the process of generating transgenic animal models. Different culture methods for ES cell exist and are constantly optimized in order to keep the cells in totipotent, undifferentiated state for gene modification and keep their germline potency. For this purpose cells were grown the on a feeder cell layer, usually mitotically inactivated murine embryonic fibroblasts [START_REF] Heuer | Development of an in vitro embryotoxicity test using murine embryonic stem cell cultures[END_REF]. The discovery of the cytokine myeloid leukemia inhibition factor (LIF) contributed to a big advancement of ES cell culturing techniques [START_REF] Schöler | A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor[END_REF][START_REF] Williams | Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells[END_REF]. LIF-mediated signaling via activation of STAT3 blocks cellular differentiation (Burdon et al., 1999) . Using LIF containing, feeder free systems for culturing ES cells that use a support matrix like gelatin or Matrigel® have been widely established. In order to maintain a homogenous and undifferentiated population, we used 0.1% gelatin and LIF in serum-containing medium. We supplemented the ES cell culture media with β-mercaptoethanol and nonessential amino acids that were reported to provide further refinement to maintain the viability and totipotency of the cultured stem cells [START_REF] Limaye | Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production[END_REF]. The introduction of the targeting vector was done by electroporation, which is the most preferred method for introducing exogenous DNA into ES cells, although not the most efficient one. In order to select for successful recombinants, cells were grown on geneticin (G418), a toxin which only allowed the growth of cells expressing the neo-gene from the targeting cassette, an enzyme that inactivates neomycin and its structural relatives such as G418. Once the positive clones are identified and verified the neo cassette was no longer necessary and was excised. This could be done directly in the ES cells by using the recombinant Cre-recombinase, but this method requires another round of transfection, which often greatly diminishes the germline potential of the ES cells. Alternatively, the neo cassette can be removed in the mouse after successful germline transmission. We applied the latter method for the generation of Dmd EGFP reporter mice by crossing the F1 generation with ubiquitous Cre-deleter mice.

The event of homologous recombination in ES cells is infrequent rare event and usually large numbers of clones have to be screened for correct recombination. The screening of positive ES cells was done by Southern blot analysis, which is the more precise method to distinguish between the wildtype and the modified allele. One can also use PCR-based screening of positive clones, which is a more rapid process but has a larger degree of false positive and false negative results. If the recombination frequency is low, it is possible that the PCR analysis will not detect the clones. If applying Southern blot analysis, the recombination has to be verified for both homology arms via a 5' and 3' specific probes that bind to regions outside of the targeting cassette. The strategy we used is represented in Figure 12. Since we were targeting the dystrophin gene located on the X-chromosome, male ES cells were used for homologous recombination ideally resulting in only one band being visible on the Southern blot. In Figure 12B on the first blot BglII digestion and 5'-probe hybridization yielded 10 positive clones. 7 of them were further tested using the same enzyme and 3' probe or BamHI Finally, the successfully generated F1-generation of Dmd EGFP-neo mice still bearing the selection cassette were analyzed via PCR and Southern blot and crossed with ubiquitous Cre-deleter mice to generate the Dmd EGFP reporter mice. The reporter mice were born at Mendelian ratios and their genotypes were determined from tail biopsies using a triple primer PCR that enabled us to distinguish between wildtype, hetero-/homozygous female and hemizygous male mice.

Characterization of the Dmd EGFP reporter mice

The reporter mice were analyzed and characterized to ensure proper expression of the dystrophin-EGFP fusion protein throughout different tissues. We were able to image dystrophin in situ based on its fluorescence signal in tissue sections as well as in single fibers. Furthermore, we wanted to find out, whether an EGFP signal could be detected in vitro in myotubes generated from differentiated satellite cells.

Dystrophin-EGFP is expressed at the sarcolemma of skeletal muscle

As already mentioned above, a C-terminal tag appended to the endogenous dystrophin might influence its folding and localization at the sarcolemma or might disturb the function of the protein. We therefore set out to investigate the muscle for correct localization of the dystrophin and is binding partners as well as to search for dystrophic features. In muscular dystrophy research the tibialis anterior (TA) muscle of the mdx mouse was frequently used as a representative muscle for investigation. Hence we decided to preferably use this particular muscle in our experiments as well (i) to make sure that our experiments are comparable to previous findings and (ii) to be able to investigate a mixed muscle with oxidative and glycolytic portions.

The expression of dystrophin-EGFP was confirmed by Western blot analysis of proteins extracted from TA muscles that displayed the correct molecular size (Figure 18). We used Cterminal (Dys2) and rod domain specific (Dys1) anti-dystrophin antibodies, which detected the fusion protein on Western blot at the same molecular size as the wildtype 427 kDa fulllength dystrophin. The addition of the FLAG-EGFP tag would generate a 454 kDa fusion protein, but on a Western blot such small a difference could not be detected in the high MW range.

The expression levels we observed in the Dmd EGFP samples appeared to be somewhat lower when using the C-terminal Dys2 antibody. A possible explanation could be the reduction of binding affinity of the Dys2 antibody through competition with the FLAG-EGFP-tag. A similar observation was made in the Western blot analysis of brain samples, where we used another C-terminal anti-dystrophin antibody (H4), the results of which will be discussed in more detail in section 8.2.4. The equal staining intensities when using the rod-domain specific antibody (MANDYS19) let us conclude the differences seen with antibodies against the C-terminus are most likely the effect of steric hindrance.

Our goal was to generate a fusion protein that would be traceable using fluorescence microscopy without the need for ex vivo antibody staining. In skeletal muscle we verified that the native EGFP fluorescence was strong enough to identify the dystrophin at the sarcolemma. Signals from immunofluorescent staining with C-terminal and rod-domain specific dystrophin antibodies nicely co-localized with the proper EGFP fluorescent signal. The anti-dystrophin rod domain specific antibody (MANDYS19) recognizes and binds to the dystrophin amino acid sequences encoded by exons 20 and 21 [START_REF] Man N Thi | Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin[END_REF]. The Dys2 antibody recognizes the last 17 amino acids of human full-length dystrophin, which differs in two amino acid positions from the murine one. The cross sections of soleus, tibialis anterior and gastrocnemius muscles showed an exact superposition of the EGFP fluorescence with the MANDYS19 immune signal suggesting the correct expression of the skeletal muscle full-length dystrophin isoform. All the alternative dystrophin promoters that control the expression of shorter dystrophin isoforms are located downstream of intron 29 and would not be picked up by the MANDYS19 antibody. Hence, the signal from the MANDYS19 antibody, which was similar to the signal of the Dys2 C-terminal antibody, confirmed tagging of the skeletal muscle Dp427 isoform and its correct localization. Furthermore, we additionally verified the sarcolemmal expression of dystrophin using the Dys2 and Dys1 antibodies via immunohistochemistry in cryosections of the gastrocnemius, soleus, tibialis anterior, quadriceps and extensor digitorum longus muscles (Appendix, 13.4, Figure A3). Dystrophin, as part of the DAPC at the sarcolemma, provides a link between the actin cytoskeleton in the cytosol and the extracellular matrix (ECM). Therefore, we also investigated the expression of two major players, laminin in the ECM and the cytoplasmic βspectrin. Laminins are heterotrimeric extracellular matrix glycoproteins. In the muscle different laminin isoforms are expressed during myogenesis, as well as at the myotendinous and the neuromuscular junction (NMJ). Moreover, they play a role in synaptogenesis [START_REF] Patton | Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice[END_REF]. In adult skeletal muscle the most abundant isoform is laminin-211 (α2/β1/γ1, previously called merosin) [START_REF] Ehrig | Merosin, a tissue-specific basement membrane protein, is a laminin-like protein[END_REF][START_REF] Sasaki | Expression and distribution of laminin alpha1 and alpha2 chains in embryonic and adult mouse tissues: an immunochemical approach[END_REF]. Laminin-211 interacts with two integral sarcolemmal proteins, with (i) integrin α7β1 and with (ii) dystroglycan, which is the core-component of the DAPC. α-Dystroglycan is heavy glycosylated, which is essential for the binding of its extracellular domains to laminin-211 and to other ECM proteins [START_REF] Yoshida-Moriguchi | O-mannosyl phosphorylation of alphadystroglycan is required for laminin binding[END_REF]. Hence, a link between extracellular laminin to intracellular actin is ensured by a non-covalent binding of α-dystroglycan to β-dystroglycan, which interacts with the C-terminus of dystrophin. In turn dystrophin completes the linking chain through its interaction with the cytoskeletal actin [START_REF] Ervasti | A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin[END_REF]). An immunofluorescent staining for laminin of the skeletal muscles is represented in Figure 19D. The distinct locations of dystrophin and laminin expression can be clearly seen with the dystrophin-EGFP signal located towards the interior of the fiber. In addition, laminin stained the blood vessels in the interstitial region between the fibers where no EGFP was detected. Spectrin, like dystrophin, belongs to the spectrin protein superfamily of actin binding, membrane-anchoring proteins that are responsible for the maintenance of membrane integrity and cytoskeletal structure. Multiple spectrin isoforms exist, which are regulated in a complex, tissue-and time-specific manner in a variety of cells [START_REF] Winkelmann | Beta spectrin in human skeletal muscle. Tissue-specific differential processing of 3' beta spectrin pre-mRNA generates a beta spectrin isoform with a unique carboxyl terminus[END_REF]. In skeletal muscle, spectrin is a component of the membrane associated cytoskeleton, and the predominant isoform is β-spectrin. It has been localized to the "costameres", which are riblike structures that lie on the cytoplasmic surface over the Z and M lines of adjacent sarcomeres [START_REF] Craig | Gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma attachment sites[END_REF][START_REF] Pardo | A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements ('costameres') mark sites of attachment between myofibrils and sarcolemma[END_REF][START_REF] Porter | Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle[END_REF][START_REF] Porter | Two populations of β-spectrin in rat skeletal muscle[END_REF]. At the costameres β-spectrin co-localizes with dystrophin [START_REF] Porter | Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle[END_REF][START_REF] Porter | Two populations of β-spectrin in rat skeletal muscle[END_REF]. In the Dmd EGFP reporter mice this co-localization was maintained thus further confirming the correct expression and co-localization of the membrane associated cytoskeleton in the reporter mice (Figure 19C). Interestingly, the endothelial cells of the endomysial capillaries were also stained by the anti-β-spectrin antibody, whereas dystrophin-EGFP expression was absent. This shows that the expression of dystrophin in the vascular system is confined to those larger vessels that possess a tunica media (e.g. arteries and larger veins) [START_REF] Rivier | Different utrophin and dystrophin properties related to their vascular smooth muscle distributions[END_REF].

Tagging of the endogenous dystrophin does not induce a dystrophic phenotype and does not disturb DAPC assembly in skeletal muscle of Dmd EGFP reporter mice

Although we were able to demonstrate that the dystrophin-EGFP protein was correctly localized at the sarcolemma, it remained to be shown that the DAPC complex was correctly expressed and localized as well. Furthermore, we wanted to make sure that no changes in muscle morphology could be observed and hence no dystrophic phenotype was induced.

In addition, we screened different skeletal muscles for the expression of representative members of the DAPC sub-complexes, the sarcoglycan-complex, the dystroglycan-complex as well as the cytoplasmic nNOS. (Figure 26, Appendix 13.4, Figure A2). Normal sarcolemmal expression patterns for α-, β-, γ-sarcoglycan, α-dystroglycan, and nNOS were confirmed suggesting proper function of the fusion protein and no obvious changes in the protein structure that might have disturbed those interactions.

In some of the cryo-cross sections of the TA the fiber in Dmd EGFP reporter mice appeared smaller than in the corresponding wildtype animals (Figure 26). This might be due to the fact that images taken were from different regions of the TA. The deep part, which is closer to the bone, contains generally fibers with smaller cross sectional areas, which increase towards the superficial region of the muscle [START_REF] Chiyoko | The Response of Tibialis Anterior Muscle Fibers in Mice to IncreasedMuscle Activity[END_REF]. However, to rule out a systematic difference in fiber diameters, a morphometric analysis of complete TA cross sections at different levels of the muscle has to be done.

Although we have verified the correct localization of the DAPC members, it remains to be shown whether the dystrophin-EGFP expression influences the amount of the other proteins in the complex expressed. Reduced levels of DAPC members have been observed in dystrophic muscles in mdx mice and DMD patients. However, quantification of protein expression or localization is challenging. Such investigations could be done by semiquantitative Western blot, which would, however, only pick up large differences. The limitations of Western blot could be overcome by mass spectrometry, especially by the SILAC method [START_REF] Ong | Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics[END_REF], which would however require a specific breeding program. As an alternative, we could exploit the presence of the FLAG or the EGFP tag in our dystrophin of Dmd EGFP reporter mice to perform an immunoprecipitation. The group of Montanaro has optimized a simple and rapid protocol for antibody-based purification of dystroglycan and interacting proteins directly from skeletal muscle tissues followed by direct protein identification by tandem mass spectrometry. This method could thus be applied to precipitate the Dystrophin-EGFP and subsequently identify and quantify the co-precipitated binding partners [START_REF] Yoon | Comparative proteomic profiling of dystroglycanassociated proteins in wild type, mdx and Galgt2 transgenic mouse skeletal muscle[END_REF].

Dystrophin-EGFP fusion protein is expressed in the heart and the smooth muscles

An important consideration for our targeting strategy was the site of EGFP insertion into the endogenous dystrophin. The aim was to target the major dystrophin isoforms expressed in different tissues and to find out whether the endogenous fluorescent signal would be strong enough for detection.

The absence of dystrophin in the heart leads to dilated cardiomyopathy, the most common cause of death in DMD patients. This underlines the importance to understand the function of dystrophin in the heart. Up to date, very few animal models exist that aim to address this question [START_REF] Bostick | Cardiac expression of a mini-dystrophin that normalizes skeletal muscle force only partially restores heart function in aged Mdx mice[END_REF]. In the Dmd EGFP mouse we observed clear dystrophin-EGFP expression in the heart and co-localization with the dystrophin immune signal, which suggests the proper expression of the Dp427 isoform (Figure 20). The dystrophin expression in the heart is controlled by the muscle-and brain-specific promoters. Moreover, intramuscular blood vessels in the heart sections showed an EGFP-signal as well, which partially co-localized with the anti-CD31 signal (Figure 20). Dystrophin is known to be expressed in the walls of large arteries, large veins and small arteries [START_REF] Rivier | Different utrophin and dystrophin properties related to their vascular smooth muscle distributions[END_REF]. This expression pattern hints towards a mechanical role of dystrophin in the membrane of smooth muscle cells since these arterioles have contractile properties and actively participate in the regulation of vascular tone and diameter as well as of blood pressure, while dystrophin is absent from small veins, whose diameter is only passively regulated by the blood volume and elastic fibers [START_REF] Rivier | Different utrophin and dystrophin properties related to their vascular smooth muscle distributions[END_REF].

We confirmed that the smooth muscle isoform in the walls of the blood vessels was successfully targeted in the cardiac tissue as well. The successful tagging and the strong EGFPsignal observed in the heart will facilitate further studies on dystrophin expression in the heart as well as in the vascular smooth muscles. In future experiments we now have to address the question, whether in vitro cultured and differentiated cardiomyocytes from Dmd EGFP reporter mice would express the fusion protein as well.

The localization of dystrophin near the plasma membrane of smooth muscle cells via its EGFPfluorescence could be readily observed also in the cryosections of intestinal organs. In crosssections of the stomach, ileum, and colon of Dmd EGFP mice we observed bundles of longitudinal and circular layers of smooth muscle cells with dystrophin located at their findings underline the importance to develop models that will improve the capability to study of the subcellular localization of dystrophin in neurons.

Unfortunately, we were unable to detect any EGFP fluorescent signals in the Purkinje cells of the cerebellum and in the neurons of the hippocampus using natural fluorescence of the Dmd EGFP mice. It is possible that the fluorescence signal was too weak to be detected by conventional fluorescence microscopy. We used 8 µm cryosections and standard methods for tissue preparation with no prior perfusion of the mice paraformaldehyde. Perfusion has been shown to improve antibody staining in the brain, while the effect on the natural EGFP fluorescence could be diminished [START_REF] Moore | Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy[END_REF][START_REF] Nguyen | Glial scaffold required for cerebellar granule cell migration is dependent on dystroglycan function as a receptor for basement membrane proteins[END_REF][START_REF] Sekiguchi | A deficit of brain dystrophin impairs specific amygdala GABAergic transmission and enhances defensive behaviour in mice[END_REF]. Using confocal imaging or two photon microscopy on thicker brain sections/slices might also help to improve the visualization and confirm the expression of full-length dystrophin in these brain regions.

In Western blot analysis of brain lysates from Dmd EGFP mice, we used antibodies against GFP as well as against the rod-and C-terminal domain of dystrophin (Dys1 and H4, respectively).

With this method we were unable to detect any bands (Figure 22). In wildtype brain, incubation with the H4 antibody revealed a very weak band and the Dys1 antibody did not bind at all. The H4 antibody is a C-terminal rabbit polyclonal antibody that recognizes the last 11 amino acids of dystrophin. In skeletal muscle we observed a weaker signal for dystrophin in the Dmd EGFP samples when using the C-terminal Dys2 antibody as compared to wildtype samples, while staining with the rod-domain Dys1 antibody revealed similar band intensities.

Obviously, also in brain the EGFP tag causes steric hindrance against binding of the H4 and Dys2 C-terminal antibodies. This would also explain why we were unable to detect the Dp427 band in the brain of Dmd EGFP mice, where the dystrophin expression is only 10% of that in skeletal muscle. In order to show that we have tagged also the full-length isoform of brain dystrophin, we will test further antibodies and plan to perform a Western blot with protein extracts from isolated hippocampus and cerebellum, brain regions with a relatively higher neuronal dystrophin expression.

The Dp140 isoform of dystrophin is highly expressed in the fetal brain where it might have a role during early brain development. In the adult brain its expression was reported to be in the microvasculature [START_REF] Lidov | Dp140: a novel 140 kDa CNS transcript from the dystrophin locus[END_REF][START_REF] Morris | Apo-Dystrophins (DP140 and DP71) and Dystrophin-Splicing Isoforms in Developing Brain[END_REF]. The shortest Dp71 isoform of dystrophin is abundant in the adult brain and its important function was investigated using animal models like the Dp71-null mice [START_REF] Sarig | Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development[END_REF]. Dp71 is expressed around blood vessels of the brain and is detected in different marcoglia cells throughout the brain and retina like astrocytes in the cortex, Bergman glial cells in the cerebellum, radial glial cells in the hippocampus and Müller glial cell in the retina [START_REF] Blake | Different Dystrophin-like Complexes Are Expressed in Neurons and Glia[END_REF][START_REF] Dalloz | Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina[END_REF][START_REF] Enger | Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse[END_REF][START_REF] Haenggi | The role of utrophin and Dp71 for assembly of different dystrophin-associated protein complexes (DPCS) in the choroid plexus and microvasculature of the brain[END_REF]. The expression of Dp71 is confined to the glial-vascular endfeet, suggesting a role in blood-brain as well as the blood-retina barrier.

In the brain and retina of Dmd EGFP mice we observed natural dystrophin-EGFP expression around blood vessels that were stained with the anti-CD31 endothelial cell marker (Figure 23, Figure 24). Moreover, staining with an antibody against the glial fibrillary acidic protein (GFAP) reveled partial co-localization of the dystrophin-EGFP with the Bergmann glia in the cerebellum, the glial cells of the hippocampus, and the Müller cells at the inner limiting membrane of the retina (Figure 23B, Figure 24C). In these regions the EGFP signal again only co-localized with the glial endfeet, which confirms a characteristic expression pattern of the Dp71 isoform [START_REF] Enger | Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse[END_REF]. On the other hand, dystrophin is expressed also in the blood vessels, mainly in the wall surrounding the endothelial cells. The EGFP signal was at some locations exactly surrounding the CD31 immunosignal, suggesting expression of the dystrophin in the outer walls of the vessels but not in the vascular endothelium. However, from our immunofluorescent images it did not become entirely clear, whether the green fluorescence really derived from the vessel wall or from the glial endfeet that constitute part of the blood-brain barrier. Additional stainings as well as improved high-resolution light imaging techniques (e.g. STED or STORM microscopy) will help to solve this question. Furthermore, Western blot analysis of brain lysates from transgenic mice using the anti-GFP antibody showed a strong band migrating at approximately 100 kDa. This band corresponds to the Dp71 isoform with an appended EGFP tag of 27.9 kDa. The anti-dystrophin C-terminal H4 antibody, which was binding to the Dp71 isoform in wildtype samples did not detect any band in the transgenic brain lysates. This observation further verifies our assumption that H4 binding is impeded by the EGFP tag and could explain why we did not detect the Dp427 band in the brain of the reporter mice. Using the Dmd EGFP reporter mice the complex role of dystrophin isoforms at the glial-vascular interface and their role in brain vascularization and blood brain barrier modulation could be further studied and better characterized. It is yet not clear how alterations in blood brain barrier observed in Dp71 null mice emerged. A direct link to the function of Dp71 in water homeostasis and potassium buffering has not been made) [START_REF] Nicchia | Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complex[END_REF][START_REF] Vajda | Delayed onset of brain edema and mislocalization of aquaporin-4 in dystrophin-null transgenic mice[END_REF]. Alternations of the blood brain barrier in mdx mice are better characterized. Loss of Dp427 leads to changes in vascular permeability, injury of endothelial cells, and disturbances of tight junctions and AQP4 assembly in glial cells [START_REF] Nico | Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice[END_REF]. These observations were explained by activation of the hypoxia inducible factor-1α together with upregulation of vascular growth factors and rearrangement of ZO-1 and claudin-1 proteins of the tight junctions [START_REF] Nico | HIF activation and VEGF overexpression are coupled with ZO-1 up-phosphorylation in the brain of dystrophic mdx mouse[END_REF]. Still a more detailed analysis need to be performed in order to understand these complex processes in the brain.

Dystrophin plays also an important role in the retina. The photoreceptors (rods and cones) of the retina are located at the outermost region called outer nuclear layer. At the outer plexiform layer (OPL) the terminals of the rods and cones interact with the postsynaptic bipolar and horizontal cells. In this region expression of dystrophin Dp427 and Dp260 have been associated with the photoreceptor terminals [START_REF] Dalloz | Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina[END_REF][START_REF] Howard | Localization of dystrophin isoform Dp71 to the inner limiting membrane of the retina suggests a unique functional contribution of Dp71 in the retina[END_REF]. In our Dmd EGFP mice the natural EGFP fluorescence could be detected in the OPL with a punctuate distribution as previously described. The signal co-localized with the immunosignal from the Dys2 antibody. Using the rod domain MANDYS19 antibody, dystrophin superposition with the EGFP signal was detected only in some regions. This finding confirms the targeting of the fulllength dystrophin as well as of the Dp260 isoform, which is not recognized by MANDYS19 (Figure 24B). In this region blood vessels stained with the anti-CD31 antibody and associated with the natural EGFP signal and with Dys2 staining, but not with MANDYS19 staining, reflect the expression of Dp71-EGFP in the endfeet of the astrocytes, at their contact with the capillary basal lamina. Dystrophin expression in the retinal blood vessels needs to be confirmed using improved imaging high resolution light imaging techniques.

As already mentioned in the introduction (sections 4.4.6.2), retinal abnormalities measured via the electroretinogram (ERG) have been observed in DMD patients as well as in different animal models [START_REF] Pillers | Dystrophin expression in the human retina is required for normal function as defined by electroretinography[END_REF][START_REF] Schmitz | Dystrophin in the retina[END_REF]. In mice that lack the fulllength dystrophin isoform, no changes in the ERG were observed, but changes appeared, when deleting the shorter isoform [START_REF] Pillers | Effects of Dystrophin Isoforms on Signal Transduction through Neural Retina: Genotype-Phenotype Analysis of Duchenne Muscular Dystrophy Mouse Mutants[END_REF]. The changes in the b-wave, observed in under scotopic conditions, measures signals from the inner nuclear layer, suggesting alterations at the synapses between the rod photoreceptors and the ON bipolar cells. At the inner nuclear layer bipolar and horizontal cells receive signals from the photo-terminals. The synapses are located in the OPL, where dystrophin expression was attributed to the photoreceptors. However, it remains to be shown whether the b-wave changes at the synapse are caused by pre-or postsynaptical changes.

Recently, authors performed more detailed in depth analysis of the dystrophin isoform distribution in the OPL of the retina using a combination of transcriptomic analysis and immunostaining. Dystrophin mRNA expression was detected at the inner nuclear layer. Using immunofluorescence, dystrophin expression was found also in the post-synapse of ON rod and cone bipolar cells, although at lower levels than in the surrounding photoreceptor elements, whereby the authors could not exclude artifacts through fluorescent signals from neighboring regions [START_REF] Wersinger | Reevaluation of Dystrophin Localization in the Mouse Retina[END_REF]. Thus using the Dmd EGFP mice a better characterization of this region can be performed without the use of antibodies and potential associated staining artifacts.

On the other hand, Müller cell mediated signaling has been proposed to contribute to b-wave generation which suggests a role for Dp71 in the ERG changes observed [START_REF] Dalloz | Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina[END_REF][START_REF] Newman | Model of electroretinogram b-wave generation: a test of the K+ hypothesis[END_REF]. Further evidence is necessary to prove this. Finally, the color blindness observed in some DMD patients [START_REF] Costa | Red-Green Color Vision Impairment in Duchenne Muscular Dystrophy[END_REF] requires better characterization of dystrophin in cone signaling. Hence, the function and distribution of dystrophin isoforms in the retina remain to be better characterized in order to understand the visual impairment observed in DMD patients.

Dystrophin-EGFP expression in single fibers and in myotubes in vitro

We were interested to see, whether the EGFP signal would be strong enough, even on the single myofiber level, to distinguish between the wildtype and the Dmd EGFP mouse. Indeed, in fixed as well as in living single fibers (floating culture, not shown) the EGFP fluorescence was strong enough to distinguish between the two genotypes (Figure 27A). Moreover, a clear enrichment of EGFP fluorescence could be observed around the NMJ. We show co-localization of dystrophin with acetylcholine receptors via staining of the fibers with α-bungarotoxin (BTX). Fortunately, the natural EGFP signal was much stronger on the single fibers that were not processed for immune staining (Figure 27,compare B and C). This observation confirms that our Dmd EGFP model can be used to study the in vivo dynamics of dystrophin at the NMJ based on its natural EGFP signal. NMJ alterations have been observed in dystrophin deficient mdx mice where the lack of dystrophin and subsequently also of members from the DAPC cause morphologic changes of the motor endplate and possibly also of synaptic transmission [START_REF] Kitaoka | Topographic comparison of subneural apparatuses at neuromuscular junctions in normal and dystrophic [mdx) mice: a scanning electron microscope study[END_REF][START_REF] Lyons | Structure and function of the neuromuscular junction in young adult mdx mice[END_REF]. Changes at the postsynaptic membrane have been mostly attributed to the lack of dystrophin via a disturbance in the distribution of acetylcholine receptors [START_REF] Shiao | Defects in neuromuscular junction structure in dystrophic muscle are corrected by expression of a NOS transgene in dystrophin-deficient muscles, but not in muscles lacking α-and β1-syntrophins[END_REF].

Another line of investigation we followed was to find out, whether ex vivo isolated satellite cells growing in vitro would express dystrophin once they form myotubes in culture.

Unfortunately, the native EGFP signal in the myotubes after 8 days of culture was too weak to be picked up by fluorescence microscopy, possibly due to the still low absolute number of dystrophin molecules available. Interestingly, using a well-established anti-GFP antibody, the dystrophin-EGFP fusion protein was detected with a much stronger signal then using any of the anti-dystrophin antibodies. This further confirms the usefulness of our animal model to study dystrophin expression during myogenesis. The ability of ex vivo satellite cells from Dmd EGFP reporter mice to produce dystrophin-EGFP provides a valuable tool to be used for transplantation and cell-tracing experiments.

Dmd EGFP reporter mice: which dystrophin isoforms express the fluorescent tag?

The aim of our study was to generate a reporter mouse, in which dystrophin expression can be visualized and traced in different tissues by means of EGFP fluorescence. Dystrophin and its numerous isoforms and splice variants have pleiotropic functions throughout the body. Almost all dystrophin isoforms share the C-terminal domain, which is very important for the function of the protein. It includes the dystroglycan, syntrophin and dystrobrevin binding sites. In two isoforms, Dp71c and Dp71Δ110, the syntrophin binding domain is removed. This isoforms are expressed during neural development in humans [START_REF] Ceccarini | A splice variant of Dp71 lacking the syntrophin binding site is expressed in early stages of human neural development[END_REF]. In mice expression of these embryonic variants could be only confirmed on the mRNA level [START_REF] Bies | Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development[END_REF].

Dmd EGFP reporter mice express EGFP fused to the last exon 79 of dystrophin. Theoretically all isoforms expressing exon 79 should be tagged. These include: Dp427 (M, B, P), Dp260, Dp140, Dp116, Dp71d and Dp71c. The alternatively spliced hydrophobic C-terminus observed in Dp71f and Dp71Δ110 cannot be targeted in our model since skipping of exon 78 shifts the reading frame of the last exon 79. In humans these C-terminally altered isoforms were shown to be involved in early embryonic development, but are also expressed in adult brain, albeit to a lesser extent [START_REF] Austin | Cloning and characterization of alternatively spliced isoforms of Dp71[END_REF][START_REF] Austin | Expression and synthesis of alternatively spliced variants of Dp71 in adult human brain[END_REF][START_REF] Feener | Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus[END_REF]. In mice, these isoforms have only recently been identified on the protein level in the adult mouse brain [START_REF] Daoud | Analysis of Dp71 contribution in the severity of mental retardation through comparison of Duchenne and Becker patients differing by mutation consequences on Dp71 expression[END_REF].

Figure 29 shows a scheme of the different dystrophin isoforms, their theoretical tagging with EGFP and the isoforms we confirmed to be expressed in the reporter mice.

We observed EGFP tagged full-length dystrophin in skeletal, heart and smooth muscle as well as in the retina. These isoforms are transcribed from the muscle and brain specific promoters.

The expression of full-length dystrophin in the brain is driven by the Brain and Purkinje promoter. Unfortunately, using EGFP fluorescence and Western blot analysis, we were not able to detect this Dp427 isoform. No EGFP signal was detected in neurons in the cerebellum or hippocampus or cortex of Dmd EGFP mice, regions where Dp427 expression was reported previously [START_REF] Huard | Localization of dystrophin in the Purkinje cells of normal mice[END_REF][START_REF] Jancsik | Differential distribution of dystrophin in postsynaptic densities of spine synapses[END_REF]. Most likely the methods we used to investigate the Dp427 expression in the brain were not sensitive enough. Experiments are on the way to show its expression in the reporter mice.

The tagging of the retinal isoform Dp260 was successfully identified by natural EGFP signal in the outer plexiform layer of the retina, where it is expressed next to the Dp427 dystrophin isoform.

Furthermore, we confirmed the successful targeting of Dp71 (i) by Western blot analysis as well as (ii) by immunofluorescence in the brain and retina. Dp71 is expressed in glia cells at the glial-vascular interface, surrounding the endothelial cells. Using fluorescence microscopy, we detected the EGFP signal at the endfeet of the Bergman glia in the cerebellum, at the endfeet of the astrocytes of the hippocampus, and in the Müller glia of the retina.

The targeting of Dp116 and Dp140 isoforms remains to be analyzed.

Future applications of the Dmd EGFP reporter mouse

In this work we investigated the main features of the Dmd EGFP model, confirming the correct expression of the dystrophin-EGFP fusion protein at known tissues and sites throughout the murine body. The model we have developed can now be applied to answer biological questions concerning the expression, localization as well as the function of dystrophin, which seem to be more diverse than initially thought. Several questions could be addressed using

Dystrophin expression during mouse development

Dystrophin plays an important role during development and the expression of different specific isoforms has been reported. Their precise function still remains to be clarified. In humans, the Dp140 and Dp71 isoforms were shown to be expressed during development and neurogenesis. In DMD patients, mutations affecting these isoforms have been linked to cognitive impairment and mental retardation [START_REF] Lidov | Dp140: a novel 140 kDa CNS transcript from the dystrophin locus[END_REF][START_REF] Morris | Apo-Dystrophins (DP140 and DP71) and Dystrophin-Splicing Isoforms in Developing Brain[END_REF]. This underlines the importance of a better characterization of their role. Furthermore, a role of dystrophin during myogenesis has been postulated in studies conducted in mdx mice. It has been shown that dystrophin would be essential for correct fiber-type specification and stem cell function [START_REF] Merrick | Muscular dystrophy begins early in embryonic development deriving from stem cell loss and disrupted skeletal muscle formation[END_REF]. However, the native spacial and temporal expression pattern of dystrophin during development remains to be determined. This question could be addressed using the Dmd EGFP reporter mice. First, however, we would have to confirm whether our model was suitable to study the expression of dystrophin based on its natural EGFP fluorescence during embryogenesis. If this is the case one could use livecell confocal microscopy of the developing embryo. Another new exiting approach was developed allowing the observation of the normal development of mouse embryos between E6.5 and E8.5 for up to 24 h using light-sheet microscopy [START_REF] Lopez | Live confocal microscopy of the developing mouse embryonic yolk sac vasculature[END_REF][START_REF] Udan | Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy[END_REF].

Dystrophin expression in adult muscle and non-muscle tissue

The subsarcolemmal expression of dystrophin in muscle tissue was clearly seen in the Dmd EGFP reporter mice. Yet, a more "mobile" dystrophin population has been recently observed in the muscle cells of zebrafish [START_REF] Bajanca | In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis[END_REF]. Whether such a population exists in mice could be analyzed using our model in combination with FRET and two-photon confocal live-cell imaging techniques as described by the group of Bajanca.

In non-muscle tissues, we observed strong EGFP expression in the photoreceptor terminals of the retina. In this region, the distribution of Dp427/Dp260 isoforms are not clearly defined.

Moreover, dystrophin was suggested to be expressed in ON bipolar cells [START_REF] Wersinger | Reevaluation of Dystrophin Localization in the Mouse Retina[END_REF]. The Dmd EGFP reporter mice are suitable to address these questions using advanced imaging techniques.

The strong EGFP signal of Dp71, which we observed at the glial-vascular interface in the brain and retina of the Dmd EGFP mice, could be exploited to study the role and dynamics of dystrophin at the blood-brain barrier and blood-retina barrier. In combination with advanced super resolution imaging techniques like STORM and PALM the expression of dystrophin could be better characterized at the subcellular or even compartmental level.

Finally, the expression of dystrophin mRNA coding for different dystrophin isoforms has been confirmed in non-muscle tissues i.e. lung, liver, kidney, thymus, spleen, testis, and uterus [START_REF] Tokarz | Redefinition of dystrophin isoform distribution in mouse tissue by RT-PCR implies role in nonmuscle manifestations of duchenne muscular dystrophy[END_REF]. The dystrophin EGFP fluorescence in the Dmd EGFP reporter mice will facilitate the investigation of the dystrophin protein expression in these organs and help to find out whether isoform-specific mRNA copy numbers really reflect the magnitude of expression of the respective isoforms.

Dystrophin expression in satellite cells

It has recently been discovered that dystrophin plays a role in the regulation of asymmetric division and polarity of satellite cells [START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF]. This opens a whole new avenue of research into dystrophinopathies. Natural EGFP expression on activated satellite cells, from single fibers should first be confirmed in our model, since the expression could be weak and below background fluorescence. If the EGFP expression was strong enough, live-cell imaging of freshly isolated single fibers with their attached satellite cells could be done in order to follow up on the exact mechanism of dystrophin expression during asymmetric division. Furthermore, we could characterize the satellite cell population expressing dystrophin by inducing injury via cardiotoxin in the Dmd EGFP mice in order to activate the satellite cells followed by FACS sorting of the EGFP expressing population. Subsequently, RNA sequencing as well as functional experiments with the cells could be performed. Furthermore, Dmd EGFP reporter mice could be cardiotoxin-injured and the muscles screened by live-cell imaging for the expression of dystrophin-EGFP in activated satellite cells.

Studying satellite cell function

Transplantation experiments using satellite cells from the Dmd EGFP mice could be conducted to understand the decline in functions of satellite cells with age and their role in sarcopenia as well as muscle degeneration after acute or chronic injury. Many factors contributing to satellite cell exhaustion during aging and dystrophic process are still not well defined. The ability of young versus old satellite cells to proliferate and repair a dystrophic or injured muscle could be studied. This would help to elucidate the contribution of cues from a young versus an aged satellite cell niche in the regenerative process. Transplanted cells could be easily traced by their EGFP fluorescence.

Studying dystrophin positive revertant fibers

An important topic to be investigated with the Dmd EGFP mouse, and the initial idea for creating this model, will be the enigma of dystrophin positive revertant fibers. The revertant fibers have been discovered in the late 80ies shortly after the DMD gene was found and after the first antibodies had been produced.

To generate such a disease model, the Dmd EGFP allele has first to be crossed into the mdx background in cis. This aim could be achieved through the natural recombination in female F1 mice that are heterozygous for the Dmd EGFP as well as the mdx allele. Crossing over and recombination during meiosis between Dmd exon 23 bearing the mdx nonsense mutation on one and the exon 79-EGFP sequence on the other X chromosome corresponds to a hypothetic recombination frequency of ≈1 centiMorgan (cM) (≈1%), since both exons are separated by ≈1 Mio bp. For this, one would expect one crossing over in ≈100 litters. A successful crossing over and the position of both mdx mutation and EGFP-tag in cis could then be detected in the male progeny of the F2 generation by genotyping. If the exon 23 mdx nonsense mutation and the EGFP-tag are on one allele in cis in a male mouse, those mice will not express the EGFPtag due to the intervening premature termination codon, at least not the full-length muscle isoform. Only in revertant dystrophin positive fibers the EGFP tag will be (re)expressed if the reading frame of exon 79 is maintained or reconstituted. Usually regions in the central rod domain surrounding the mutation are skipped in those fibers and the last exon 79 would be present [START_REF] Lu | Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion[END_REF]. The Dmd mdx-EGFP model will facilitate the study of dystrophin positive revertant fibers and will enable their tracing as well as extraction and analyses of their attached satellite cells.

In preparation for such an experiment, we were able to show that EGFP expression on the level of single fibers can be clearly distinguished from non-EGFP expressing fibers. These results suggest that single fibers with a dystrophin positive revertant region could be distinguished from non-revertant ones (dystrophin negative). Hence, their attached satellite cells that are located close to the region where the revertant event had taken place, could be isolated, multiplied in culture and analyzed on the mRNA or protein level. Furthermore, using laser capture microdissection an area of revertant fibers can be directly dissected from muscle cross sections (without prior staining), RNA isolated and sequenced. This experiment will give us insights into whether such revertant events occur on the genomic level (by somatic deletions) or on the mRNA level (by alternative splicing patterns). The splicing pattern can be then compared between clusters of the same muscle as well as between different muscles.

Moreover, using the RNA-seq technology other genes that are upregulated or downregulated and might play an important role in revertant fiber formation can be identified if compared to non-revertant fiber regions.

The use of Dmd EGFP reporter mice in exon skipping approaches for DMD

Once the Dmd mdx-EGFP model is established, it will be very useful to test and to improve various exon skipping approaches against DMD. Successful restoration of dystrophin expression was reported after the use of a novel antisense oligonucleotides the tricycle-DNA (tcDNA) oligomers, leading to an improvement of brain, heart, and skeletal muscle function [START_REF] Goyenvalle | Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers[END_REF]. Use of the Dmd mdx-EGFP mouse will facilitate and aid optimization experiments with tcDNA-AON since dystrophin restoration can be confirmed and analyzed in muscle and in non-muscle tissues by direct EGFP fluorescence. Measurement of the in vivo fluorescent signal will be much more accurate than using dystrophin antibodies and will enable serial investigations (time series) in the same mouse. Moreover, the Dmd EGFP mice or their ES cells could be used to introduce other dystrophin mutations via the CRISPR/Cas9 system [START_REF] Jinek | A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity[END_REF][START_REF] Wang | One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[END_REF] followed by screening of exon skipping molecules that are relevant to the treatment of characteristic human DMD deletion patterns.

In conclusion the Dmd EGFP mouse model will be a very useful tool to further analyze and understand the role of dystrophin and thus the pathology of DMD and will aid the development of exon skipping approaches.

Conclusion

In the present work, we describe the first dystrophin-reporter mouse model, in which the endogenous dystrophin protein was tagged with the fluorescent EGFP-protein. The generation and characterization of the Dmd EGFP reporter mice was described in detail and the application of the model especially in novel super resolution and live-cell imaging techniques discussed. We were able to confirm that the introduction of the EGFP-tag did not have any harmful influence on the expression, distribution and function of the native dystrophin protein. The fusion protein is expressed and can be detected in vivo based on its natural green fluorescence in skeletal muscle, heart, and smooth muscle as well as in CNS, as shown for brain and retina. The reporter mice were healthy, viable with normal litter sizes and without any changes of muscle architecture or expression of DAPC components. In the past 28 years after the discovery of the DMD gene and its protein product dystrophin many advances have been made to unravel the function of dystrophin in skeletal and heart muscle, the tissues mostly affected in DMD patients. More recently studies on the role of dystrophin in the CNS and its implication on cognitive development have emerged. Not much is known about the function of dystrophin during embryonic development or its role in smooth muscle tissue, and in non-muscle tissues such as liver, spleen, thymus, kidney, and uterus. In our reporter mouse model, dystrophin-EGFP expression can be visualized and followed on the level of single isolated muscle fibers and in satellite cell derived myotubes. The recent discovery of the function of dystrophin in the satellite cells points out that there are still unknown functions to be discovered [START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF] . Another recent study by [START_REF] Bajanca | In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis[END_REF] using a human dystrophin cDNA-reporter in the zebrafish model showed that there is a subgroup of dystrophin molecules that are more mobile within the muscle cell than others, a phenomenon not yet understood.

Our model provides a very promising experimental system to study dystrophin in a nonartificial system exploiting the natural EGFP-fluorescence produced from the natural promoters of mouse dystrophin. Dynamic processes can be investigated using recently developed imaging techniques in vivo and ex vivo that will contribute to a better understanding of the role of dystrophin in normal conditions as well as in disease states.

Crossing the Dmd EGFP reporter allele into different mdx-dystrophic backgrounds or introducing a desired mutation/deletion into the ES-cells of the Dmd EGFP reporter mice could be a way to develop a promising model to test different exon skipping approaches or to study revertant dystrophin fibers. Overall, the Dmd EGFP reporter mouse has a great potential to unravel unknown functions of dystrophin, to aid the visualization of its precise localization and to discover novel interaction partners.

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Berlin

  to cordially thank all whose support and encouragement contributed to the successful completion of my doctoral thesis. First of all, I would like to thank my supervisors Prof. Dr. M. Schuelke and Prof. Dr. H. Amthor for giving me the opportunity to work on an exciting project over the past 4.5 years. I will always appreciate their scientific advice and their work ethics. I am very grateful to Prof. Dr. M. Schuelke for his generosity, his unconditional support, his guidance and for giving me the freedom to conduct research. His interest and passion in the field of the biomedical research, and his optimism always encouraged me and contributed to the success of my work. I would like to cordially thank Prof. Dr. S. Stricker of the Freie Universitat Berlin for his agreement to review my doctoral thesis. I would like to cordially thank Prof. Dr. V. Straub and Prof. Dr. K. Patel for their willingness to review my doctoral thesis. I thank Dr. G. Butler-Browne for agreeing to be part of my thesis committee.

  Protein, welches wesentlich für den Aufbau des Dystrophin-assoziierten Proteinkomplexes (DAPC) ist. Der Komplex vernetzt das Zytoskeleton mit der extrazellulären Matrix, wodurch das Dystrophin entscheidend zur Stabilisierung des Sarkolemmas und zur Erhaltung der Muskelfaserintegrität während der Muskelkontraktion beiträgt. Mutationen im Dystrophin kodierenden DMD Gen, verursachen die schwere Xchromosomal vererbte Muskeldystrophie Duchenne (DMD). DMD ist gekennzeichnet durch eine fortschreitende Muskeldegeneration und Fibrose, welche die Skelettmuskel-und Herzmuskelfunktion beeinträchtigt. Darüber hinaus sind zu einem gewissen Grad auch kognitive, visuelle und gastrointestinale Funktionen gestört. Ursache für die Krankheit ist das Fehlen des Dystrophin-Proteins in dem jeweiligen Gewebe. In der vorliegenden Arbeit habe ich ein neues Mausmodel, die Dmd EGFP Reportermaus, generiert und charakterisiert. Das Model ist gekennzeichnet durch die Expression eines Fluoreszenz-markierten Dystrophin-EGFP Fusionsproteins. Bislang existiert keine Reportermaus für Dystrophin, so dass die Visualisierung der Dystrophin-Expression bisher nur mittels indirekter Antikörperfärbung ex vivo möglich war. Für die Herstellung der transgenen Maus habe ich einen Targeting-Vektor entwickelt, welcher die FLAG-EGFP Gensequenz downstream des letzten Exons (Exon 79) des Dmd Gens der Maus einfügt. Weiter 3' downstream habe ich noch eine loxP flankierte Neomycin Selektionskassette eingebracht, welche später nach homologer Rekombination und Blastozysteninjektion durch Verpaarung der F1-Generation mit einer ubiquitär exprimierenden Cre-Maus wieder entfernt werden konnte. Zum Nachweis der Dystrophin Expression und zum Ausschluss einer möglichen Induktion einer Dystrophinopathie durch das EGFP-markierte Fusionsprotein habe ich die Dmd EGFP Mäuse und ihre wildtyp Wurfgeschwister histologisch charakterisiert. Dabei konnte ich eine starke natürliche EGFP Fluoreszenz sowohl in den Skelett-Herz-, und glatten Muskeln, als auch im Gehirn und Auge beobachten. Das EGFP-Signal kolokalisierte mit dem immunhistologisch nachgewiesenen Expressionsmuster des Dystrophins, was die korrekte Markierung der Mehrheit der Dystrophin Isoformen bestätigt. Im Skelettmuskel wurden Dystrophin sowie andere Proteine des DAPCs in normaler Menge am Sarkolemma exprimiert. Der Muskel bewahrte seine normale Gewebestruktur, was die korrekte Funktion des Dystrophin-EGFP Fusionsproteins impliziert. Sowohl isolierte Muskelfasern als auch aus Satellitenzellen generierte Myotuben exprimierten das EGFP-Fusionsprotein in vitro. Somit ist die neue Reportermaus ein nützliches und wertvolles Model für die direkte Visualizierung der Dystrophin Expession. Darüber hinaus kann man das Model benutzen, um in oder ex vivo die Dystrophin-Reexpression nach Gentherapie oder in natürlich vorkommenden "revertant fibers" zu studieren.

Figure 1 :

 1 Figure 1: Duchenne's original case showing marked calf enlargement and lumbar lordosis. From(Emery and Muntoni, 2003, p14).

Figure 2 :

 2 Figure 2: Structure of the skeletal muscle.The skeletal muscle is attached to the bone via a tendon and composed of myofibers that are organized into fasciculi. Each myofiber, a multinucleated muscle cell, is surrounded by the plasmalemma and the basal lamina. Sandwiched between these two layers reside the skeletal muscle stem cells, also called the "satellite cells". The myofiber contains numerous myofibrils, actin and myosin filaments which are organized into the sarcomeres. The contractile units consist of actin and myosin filaments. The sarcomeres are divided into alternating I-and Abands giving the fiber their "striated" appearance. Modified from[START_REF] Relaix | Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage[END_REF]

Figure 3 :

 3 Figure 3: Organization of the DMD gene and its protein products. (A) The DMD gene is 2.5 Mb long and encodes at 7 different isoforms. The "full-length" dystrophin transcript encodes the 427 kDa isoform which is named Dp427. For this isoform, three different tissue specific promoters exist: brain (B), muscle (M), and cerebellar Purkinje cells (P). Protein products differ in their N-terminal region. Shorter dystrophin isoforms are produced from distally located promoters and expressed in the retina (Dp260); brain and kidney (Dp140); peripheral nerve (Schwann cells) (Dp116) or are expressed ubiquitously (Dp71). (B) Schematic representation of the functional domains of different dystrophin isoforms and their localization. H1-H4 hinge regions are proline rich and interrupt the rod, spectrin-like domain. The C-terminal region (CT), whichis present in all isoforms, contains binding sites for proteins of the dystrophin associated protein complex (DAPC). The cystein-rich domain includes the WW binding domain, which mediates specific protein-protein interactions with short proline-rich or proline-containing motif, the ZZ-type-zinc finger binding domain and a coiled-coil domain in the C-terminus. Modified from[START_REF] Blake | The neurobiology of duchenne muscular dystrophy: learning lessons from muscle?[END_REF].

Figure 4 :

 4 Figure 4: Dystrophin and the dystrophin associated protein complex (DAPC) in muscle tissue.Dystrophin interacts with actin through its N-terminal domain and part of the rod domain. 24 spectrin repeats compose the rod domain and are followed by a WW protein binding domain, which interacts with βdystroglycan. The latter connects to laminin at the ECM via α-dystroglycan. The dystroglycan complex at the sarcolemma interacts with the other big transmembrane complex, which is composed of sarcoglycans. Sarcospan covers the sarcoglycan complex towards the ECM. At the C-terminus dystrophin reacts via its cysteinerich domain with syntrophins that in turn can bind to α-dystrobrevin, nNOS and caveolin-3[START_REF] Davies | Molecular mechanisms of muscular dystrophies: old and new players[END_REF].

  Plastic materials were obtained from Becton Dickinson GmbH (Heidelberg, Germany), Biozym Scientific GmbH (Hessisch-Oldendorf, Germany), Braun (Melsungen, Germany), Carl Roth GmbH (Karlsruhe, Germany), Eppendorf AG (Hamburg), Falcon/Corning (BY, USA), GE Healthcare (Munich, Germany), Kisker (Steinfurt, Germany), Sarstedt AG & Co (Nürnbrecht, Germany), Thermo Fischer Scientific Inc. (Langenselbold, Germany), TPP AG (Trasadlingen, Switzerland) and VWR (Darmstadt, Germany).

  (neo) in the 3'UTR that was flanked by two loxP sites. The targeting vector was linearized with SalI and electroporated into 129Ola-derived ES cells. Correctly targeted ES clones were selected by Southern blotting via restriction enzyme sites (BglII and BamHI) that had been introduced into the vector and injected into C57BL/6 blastocysts. Chimeras were generated and mated to C57Bl/6N mice. Germline transmission of the targeted dystrophin allele was confirmed in the F1 generation of the heterozygous females via PCR screening for the neomycin cassette as well as by Southern blot analysis. The ES cell culture and generation of transgenic mice (F1 generation) have been done commercially (Polygene AG, Switzerland).

  The 10.943 kb genomic fragment spanning intron 78, exon 79 and part of the 3'UTR of the Dmd gene was subcloned from a murine Sv129-derived BAC clone (GenomeCube, bMQ389g17) via recombineering and gap repair into a pCR-Script vector (Stratagene, Holland) containing homology arms. The HAs enabled the homologous recombination and retrieval of the Dmd fragment of interest, thus generating Dmd exon 79 retrieval vector. In a second recombineering step, Dmd exon 79 was modified so that the stop codon was exchanged for a leucine (TAG>TTG) and a FLAG-EGFP sequence inserted in-frame followed by a sequence in the 3'UTR containing a loxP flanked neo cassette regulated by PGK and Em7 promoter for expression in prokaryotic and eukaryotic cells, respectively. The so generated targeting vector included also a SalI restriction enzyme site for linearization and subsequent electroporation into ES cells as well as BglII and BamHI restriction sites for Southern blot screening of positive ES clones for homologous recombination. The results of the detailed cloning steps are described in the section 7.1.1.

  strands. A heat stable DNA polymerase synthesizes the second DNA strand starting from the matrix complementary double strands (elongation). In this work, Phusion TM DNA-Polymerase (Finnzymes, Espoo, Germany) with a proofreading ability or the GoTaq TM DNA-Polymerase (Promega, Mannheim, Germany) without proofreading function were used. A standard reaction volume for the Phusion TM polymerase contained the following components: 10 µl of 5 x reaction buffer, 0.2 mM dNTPs, 0.5 µM forward and reverse primers, 3% DMSO, 1 unit DNA-polymerase, and 0.2-150 ng template DNA in a total reaction volume of 50 µl.

  counting by using counting chamberCell numbers were determined by using the Neubauer cell counting chamber. 10 μl of wellresuspended cell suspension was mixed with 1x trypan blue solution. Trypan blue stains only dead cell and the viable, unstained cell were counted. ≈10 μl the mixture was filled into the counting chamber and cells were counted. The cell number in two big squares was determined and then divided by 2 to obtain the average count in one big square (one big square corresponds to a volume of 0.1 μl), which gives the value 'counted cells' and can be replaced in the following formula: cells/ml = counted cells x F x 10 4

  Mice were kept and bred according to animal welfare guidelines under the registration of the LaGeSo (T 0222/13) in the SPF animal husbandry facility of the Charité (FEM) and of the Charité Cross Over Building. The breeding conditions were constant temperature at 22°C with humidity from 50 to 60%, 12 h light/dark rhythm, light brightness 300 lux, and enriched environment. The animals were fed with a standard pelleted diet and autoclaved drinking water. The general state of health was monitored at regular intervals by the experimenter, by animal care-takers, as well as by a veterinarian. Microbiological and virological tests for common rodent pathogens were performed at regular intervals.

  After 10 days of selection, a total of 200 clones was picked and analyzed via Southern blot analysis. After expansion and freezing of the isolated clones, DNA was prepared and digested with the restriction enzyme BglII, run by agarose gel electrophoresis and blotted onto a Hybond® membrane. A [ 31 P]radioactively labeled 5'-external probe was used to detect the proper genomic region on the X-chromosome. The primers used for generation of the probes are listed in Appendix 13.1, TableA1. After hybridization to the BglII digested genomic DNA, a fragment of 15.8 kb should be detected for the wildtype allele, whereas the targeted allele would result in a fragment of only 6.2 kb. All positive clones were further analyzed via Southern blot analysis on the 3' end for homologous recombination using a 3' external probe. The correct clones were then thawed, expanded, and DNA was prepared. For confirmation, a second Southern blot analysis was performed using the restriction enzyme BamHI and the 3' external probe. Correct 129Ola derived ES cells clones were injected into blastocysts of C57Bl/6 mice. Injected blastocysts were transferred into two CD-1 foster mice. The chimeras were mated to C57Bl/6N mice for germline transmission of the modified dystrophin locus at the X chromosome. Germline transmission was confirmed by coat color, as well as via Southern blot analysis, PCR genotyping.6.4.4. Removal of the neo-cassette using a ubiquitous Cre-deleter miceAnimals with successful germline transmission of the Dmd-FLAG-EGFP-loxP-neo-loxP construct were then crossed with a ubiquitous Cre-expressing (Cre-deleter) mouse in order to remove the loxP flanked neo cassette. The resulting Dmd EGFP mice and their wildtype littermates were subsequently used for analysis and characterization. All animals of the F1

Figure 5 :

 5 Figure 5: Flowchart for the generation of Dmd EGFP reporter mice.

Figure 6 :

 6 Figure 6: Strategy for genomic modification of the murine Dmd locus.A targeting vector was constructed for insertion of a FLAG-EGFP coding sequence into the 3'UTR of the Dmd gene so that a dystrophin-EGFP fusion protein would be expressed. The last exon 79 was modified by exchanging the stop codon to a leucine (an asterisk indicates the modified exon 79, and a FLAG-EGFP sequence with a loxP-sites flanked neo cassette was inserted. The Dmd wildtype allele was targeted in ES cells after electroporation of the SalI linearized targeting vector and subsequent homologous recombination. Dashed lines represent homologous regions. BglII and BamHI restriction sites were artificially inserted into the transgene to facilitate identification of targeted ES cells by Southern blot analysis using either 5' and 3' hybridization probes (blue squares) located outside of the targeting vector. Positive ES cells were used for generation of transgenic mice. The neo cassette was removed by crossing the F1 generation mice with ubiquitous Cre-deleter mice (constructs are not drawn to scale). Ex: exon.

Figure 7 :

 7 Figure 7: Subcloning of the 10.943 kb Dmd exon 79 genomic fragment by gap repair.Homology arms (HAs) were amplified from BAC DNA using primer pairs with the following restriction enzyme sites: Sall, NotI, and HindIII (5'HA) as well as HindIII and NotI (3'HA). HAs were cloned into the pCR-Script-AMP vector. In a second step BAC DNA was electroporated into E. coli SW106 cells. Subsequently, recombineering genes were induced and the HindIII linearized pCR-Script+HAs DNA was transformed into these cells. After successful recombination via gap-repair the 10.943 kb Dmd exon 79 fragment was cloned into the pCR-Script+HAs vector generating the Dmd exon 79-retrieval vector. Positive clones were selected by their ampicillin (Amp r ) resistance. Ex: exon, HA: homology arms.

Figure 8 :

 8 Figure 8: Generation of the PL452+mini targeting cassette.In the first step (1.) 5' and 3' mini homology arms were amplified from BAC DNA using 5'-tailed oligonucleotide primers: for the 5' mini arm spanning exon 79, KpnI and EcoRI-BglII-FLAG-modified exon 79* containing primers were designed. The 3' mini arm contained the BamHI and SacII recognition sequences. After amplification, the 200 bp mini arms together with the PL452 vector were digested and the fragments were inserted: the 5' mini arm with exon 79*-FLAG upstream of the loxP-neo cassette, and the 3' mini arm downstream of the loxP flanked neo cassette. In a second step (2.) an EGFP sequence was amplified with EcoRI flanked primers from pEGFP-N3-L221K and cloned into PL452+mini HAs downstream of the FLAG sequence generating the PL452+mini targeting cassette. A unique BglII site was included for later Southern blot analysis. Asterisk (*) indicates the modified exon 79. Ex: exon, HAs homology arms, Amp r : ampiciline resistance, NEO r : neomycine resistance.

Figure 9 :

 9 Figure 9: Generation of the targeting vector.The mini targeting cassette was digested from the PL452 vector using KpnI and SacII restriction enzymes and electroporated together with the Dmd exon 79-retrieval vector into E. coli SW106 cells, in which the recombineering genes had been induced. Homologous recombination resulted in the insertion of the mini targeting cassette into the retrieval plasmid, thereby modifying the Dmd fragment. The targeting vector can be linearized with SalI. Ex: exon, HAs homology arms, Amp r : ampiciline resistance, NEO r : neomycine resistance.

Figure 10 :

 10 Figure 10: Functionality test #1: Expression of the Dmd exon 79*-FLAG-EGFP cassette in mammalian cells. COS cells were transfected with the control plasmid Mito-RFP and the pCMV-Tag3A-Cox8a vector expressing the subcloned modified Dmd exon 79*-FLAG-EGFP cassette. One RFP positive (red) cell shows also EGFP expression (green) and both signals co-localize (merge + DAPI, yellow). The asterisk depicts the modified exon 79 with absence of the stop-codon. Scale bar = 20 µm.

Figure 11 :

 11 Figure 11: Functionality test #2: Excision of neo cassette. (A) In the presence of the neo cassette in the targeting vector primer pair#1 (S1240 and S3007) amplified a 1069 bp fragment, which would be absent after excision of the neo cassette. Primer pair#2 (S3015 and S3007) amplified a 500 bp fragment only after removal of the neo cassette. (B) Colony PCR of three clones after transformation of Cre-recombinase induced E. coli SW106 bacteria with the targeting vector using primer pair#1 (lanes 2-6) and primer pair#2 (lanes 7-11). Lane 1: 5 µl 100 bp marker; lanes 2, 3, 4 and 7, 8, 9: DNA from three positive bacterial clones after neo excision, lanes 5 and 10: PCR products from the DNA of the control targeting vector containing the intact neo cassette; lanes 6 and 11: negative water control. The three positive clones show the 500 bp band (lanes 7-9) and no band (lanes 2-4), thus confirming the excision of the neo cassette. Only from the DNA of the control targeting vector the 1069 bp fragment could be amplified verifying the presence of the neo cassette. bp: base pairs, Ex: exon.

Figure 12 :

 12 Figure 12: Detection of homologous recombination in ES cells via Southern blot analysis.(A) 5' and 3' DNA probes (blue squares) complementary to a genomic region outside of the targeting cassette were generated. In the wildtype allele the 5' and 3' probes detects a 15.9 kb band after digestion with BglII, whereas in the targeted allele a 6.2 or 12.4 kb band can be detected, respectively. Using BamHI for DNA digestion the 3' probe hybridized to an 18.5 kb fragment in the wildtype and to a 14.3 kb fragment in the targeted cells. (B) DNA from ES cell clones was digested with BglII and hybridized with the 5' probe. Ten positive clones with the recombinant allele are numbered and marked with red (left blot) and two wildtype clones were numbered and marked in black. The DNA from positive clone 2, 3, 4, 6, 7 and 11 was further hybridized with the 3' probe after BglII and BamHI digestion (right blots). As control, wildtype clone 1 and 9 (black) were tested. Clone 6 marked in red with an asterisk was the one that later led to the successful generation of chimeric mice and germ line transmission. Wildtype DNA was used as control. WT: wildtype, M: marker, kb: kilobase-pair, Ex: exon.

Figure 13 :

 13 Figure 13: Chimeric mice generated from ES cell clone 6. Germline transmission was achieved from chimera number 14 (left picture).

Figure 14 :

 14 Figure 14: Genotyping PCR and Southern blot analysis for the targeted allele. (A) PCR screening of mice from the F1 generation for the presence of the neo cassette using neo-specific primers for amplification of a 542 bp fragment. Different concentrations of the targeting vector were used as positive controls. The animals carrying numbers 1018 and 1019 were positive for the neo cassette, whereas in animal 1020 no band was detectable. (B) Southern blot analysis confirms the PCR results. DNA from tail biopsies was digested with IBglI and probed with the 5' probe. The 6.2 kb band for the targeted allele can be observed in animals 1018 and 1019. Mouse 1020 showed the wildtype band running at 15.9 kb. M: Marker, WT: wildtype, pg: picogram, kb: kilo bases, bp: base pairs.

Figure 15 :

 15 Figure 15: Genotyping PCR for the targeted dystrophin allele and neo cassette excision in transgenic mice.(A) A three primer PCR was used for genotyping. Primer S4228 binds to intron 78, primer S4269 binds to the EGFP sequence and primer S4229 to the 3'UTR. A 202 bp band is only detectable in samples from wildtype animals (S4228 and S4229), amplification using primer S4269 and S4229 will generate a 350 bp band only from the recombinant allele, after removal of the neo cassette. (B) Agarose gel electrophoresis of the genotyping PCR. Animals 1-3 had the wildtype allele and were either male (-/y) or female (-/-). In Dmd EGFP homozygous male (+/y) and female (+/+) mice the 350 bp band is detected (lanes 4, 5, 7, 8). In heterozygous females (+/-) both amplicons were detected (lane 6). Ex: exon, bp: base pair, M: Marker, WT: wildtype.

  shows a strong native EGFP fluorescent signal on cross and longitudinal sections from the tibialis anterior (TA) muscle. Moreover, staining of TA muscle sections with an anti-FLAG-tag antibody showed exact superposition of the immune signal with the natural EGFP fluorescence (Figure 17).

Figure 16 :

 16 Figure 16: Native EGFP expression in tibialis anterior (TA) muscle sections. TA cross and longitudinal sections show natural EGFP fluorescence in Dmd EGFP mice that can be detected at the sarcolemma without amplification of the signal. No fluorescent signal was observed in the wildtype (WT) cross section with the same illumination parameters. Scale bar = 50 µm.

Figure 17 :

 17 Figure 17: Co-localization of EGFP-and FLAG-tag in tibialis anterior (TA) muscle sections. TA cross sections from Dmd EGFP mice were stained with anti-FLAG (red) antibody and showed co-localization with the natural EGFP fluorescence. Sections were counterstained with DAPI (blue). Scale bar = 50 µm (left column). Higher magnification of the stained TA section is depicted in the right column. Scale bar = 10 µm.

Figure 18 :

 18 Figure 18: Western blot analysis of dystrophin-EGFP expression in tibialis anterior (TA) muscles.Western blot analysis of whole TA muscle extract using dystrophin antibodies specific to the rod domain (Dys1) and to the C-terminal domain (Dys2) reveals normal expression of the full-length protein in the Dmd EGFP mice. The anti-GFP antibody detects the tagged dystrophin protein in the transgenic animals. For control a human muscle sample was tested with the Dys2 antibody. α-Actinin2 served as loading control. WT: wildtype, ctrl: control.

Figure 19 :

 19 Figure 19: Correct localization of the EGFP-tagged dystrophin at the sarcolemma. Immunofluorescent staining of cross sections from soleus (SOL), gastrocnemius (GAS) and tibialis anterior (TA) muscles of transgenic mice with dystrophin antibodies specific to the C-terminal domain (Dys2) (A), the rod-domain (MANDYS19) (B), and antibodies specific to the cytoskeletal β-spectrin (C) and the basement membrane protein laminin (D) all colored in red. Exact co-localization was observed between the natural EGFP fluorescence (green) and the signals deriving from the two anti-dystrophin antibodies. Scale bar = 50 µm. The columns on the right side depict a higher magnification of the stained TA sections counterstained with DAPI (blue). Scale bar = 10 µm.

Figure 20 :

 20 Figure 20: EGFP expression in the heart. (A) Immunofluorescence staining of heart sections with the Dys2 antibody (red) showed co-localization of the natural EGFP signal with dystrophin in the cardiac myocytes. Scale bar = 50 µm. A higher magnification is depicted on a second column, which showed the localization of dystrophin-EGFP at the sarcolemma more clearly. Scale bar = 20 µm (B) Blood vessels in the heart were stained with anti-CD31 (red) and also showed co-localization with EGFP signal; counter stain with DAPI. Scale bar = 10 µm.

Figure 21 :

 21 Figure 21: EGFP expression in smooth muscle. (A) Sections from the stomach, duodenum, ileum and colon of Dmd EGFP mice were fixed and stained with DAPI (blue). Strong natural EGFP expression was detectable in these organs. Scale bar = 10 µm. (B) Immunofluorescence staining with an anti-dystrophin antibody (C-terminal, rabbit) (red) of ileum and colon sections verifies co-localization with natural EGFP fluorescence. Sections were counterstained with DAPI (blue). Scale bar=10 µm.

Figure 22 :

 22 Figure 22: Western blot analysis of dystrophin-EGFP expression in the brain.Western blot analysis of whole brain lysates using dystrophin antibodies specific against the rod domain (Dys1) and C-terminal domain (H4) revealed normal expression of Dp427 and Dp71 in wildtype mice, but no signal was detected in the Dmd EGFP mice. The anti-GFP antibody detected the tagged Dp71-EGFP protein in the transgenic animals with an estimated size of 100 kDa. No bands were detected using anti-Dys1 rod specific antibody. Vinculin served as loading control. WT: wildtype, kDa: Kilodalton, M: marker.

Figure 23 :

 23 Figure 23: EGFP expression in brain. (A) Immunofluorescence staining of brain sections with the Dys2 antibody shows co-localization of the natural EGFP signal with the anti-dystrophin signal in the cerebellum of the mouse. The partial co-localization of the EGFP signal with the anti-CD31 signal confirmed dystrophin expression also in the blood vessels in the brain. Scale bar = 50 µm (left column of each stain). Scale bar = 10 µm. (B) Immunofluorescence staining with anti-glial fibrillary acidic protein (GFAP) shows co-localization with the EGFP signal at the glial endfeet in the cerebellum and hippocampus. Scale bar = 50 µm in the left column of each region stained with anti-GFAP. A higher magnification is shown in the right columns. Scale bar = 10 µm.

Figure 25 :

 25 Figure 25: Dmd EGFP reporter mice show normal histopathology. (A) Haematoxylin and Eosin (H&E) stained tibialis anterior (TA) sections from adult and aged wildtype (WT) and Dmd EGFP mice showed normal morphology. H&E staining of an aged-matched mdx TA muscle section demonstrated the morphological characteristics of muscle dystrophy comprising a large variation in fiber size, mononuclear cell infiltrates, fibrosis, and abundant centrally located myonuclei. Scale bar = 20 µm.

Figure 26 :

 26 Figure 26: Dmd EGFP reporter mice showed normal histopathology. Wildtype and transgenic mice showed normal localization and expression of the DAPC components α dystroglycan, γ-sarcoglycan and nNOS in tibialis anterior (TA) cross sections. Utrophin was not up-regulated in the TA muscles of transgenic animals. Scale bar = 50 µm.

Figure 27 :

 27 Figure 27: EGFP expression of single fibers. (A) Individual fixed single fibers from EDL muscles were easily distinguished between Dmd EGFP mice and wildtype littermates based on the natural EGFP fluorescence without further amplification of the signal. Fibers were fixed and stained with DAPI. Scale bar = 50 µm. (B) Natural EGFP expression at the neuromuscular junction of single fibers stained with an acetylcholine receptor binding neurotoxin (α-Bungarotoxin, BTX, red), Scale bar = 50 µm. In the second row, higher magnification shows the co-localization of dystrophin-EGFP with the acetylcholine receptor. Scale bar = 10 µm. Fibers counterstained with DAPI. (C) Natural EGFP expression at the neuromuscular junction of single fibers, fixed and conterstained with DAPI. The NMJ without an immunofluorescent staining shows stronger EGFP signal then in the BTX stained one. Scale bar = 10 µm

  homologous recombination. For the targeting strategy, we had to consider what kind of fluorescent tag to use and at what position to insert it into the Dmd gene.

  digestion and the 3'-probe. All clones displayed the targeted allele but showed an additional signal of the wildtype band. Most likely a contamination or a mixture with DNA from wildtype cells (micro-heterogeneity) would explain the presence of the wildtype band in the samples with positive recombinant cells. An incomplete digestion by the enzyme would also yield the same result, which could have happened due to adhering DNA-binding proteins that could have obscured the respective endonuclease restriction site. Nevertheless, the positive signal verified the presence of ES cells carrying the modified allele on the X-chromosome and therefore the ES cells were used for blastocyst injection. After several rounds of injecting different positive clones, chimeric mice were generated that transmitted the knock-in allele to the F1 generation. The ES cell clone that in the end successfully achieved germline transmission of the modified Dmd allele originally had yielded two bands on the Southern blot analysis. The generation of transgenic mice took more than 2 years. The huge delay was due to problems in the ES culture of the commercial company. The process of electroporation of ES cells was repeated twice since the first round had not yielded chimeras. The ES culture methods could be the reason for the failure of germline transmission in the first round, where other factors than the supplements used to keep the cells in undifferentiated state may play an important role as well. The cell morphology and confluency are additional factors that influence the chimerism and later germline transmission, especially if the cultures are overgrown or at plateau. The concentration of the DNA construct as well as the electroporation parameters could also affect the viability of the cells. Furthermore, chromosomal abnormalities often observed in murine ES cells, may also contribute to low chimerism. This can be excluded by karyotyping of the positive ES clones and would save time on the long run.

  common and well established techniques like fluorescence, confocal, two-photon, and FRET (Fluorescence Resonance Energy Transfer) microscopy. Moreover, dystrophin-EGFP expression in the reporter mice could also be studied with a combination of novel imaging techniques. Live confocal imaging, live two-photon, light-sheet microscopy, STORM (stochastic optical reconstruction microscopy), and PALM (photoactivated localization

Figure 29 :

 29 Figure 29: Targeting of various dystrophin isoforms in Dmd EGFP reporter mice. A schematic representation of known dystrophin isoforms and splice variants and their alternative C-termini. In Dmd EGFP mice the FLAG-EGFP sequence is fused to the exon 79 coding sequence of Dmd. Theoretically we expected successful targeting of the following isoforms Dp427 (M, B, P), Dp260, Dp140, Dp116, Dp71, Dp71d and Dp71c. The alternatively spliced variants of Dp71: Dp71f and Dp71Δ110 contain an alternative C-terminus that shifts the original reading frame of exon 79 and hence cannot be tagged with EGFP. We confirmed the expression of dystrophin-EGFP fusion protein in Dp427 (M), Dp260, Dp71, but could not detect Dp427 (B, P). Light blue squares represent alternative exons at the N-terminus of some isoforms. Dark blue squares show the alternative C-terminal sequence generated by exon 78 skipping. M: Muscle specific promoter, B: brain specific promoter, P: Purkinje cell promoter, n.d.: not determined, N: N-terminus, C: C-terminus.

Figure 1 :

 1 Figure 1: Duchenne's original case ............................................................................................

Figure 2 :

 2 Figure 2: Structure of the skeletal muscle. ..............................................................................

Figure 3 :

 3 Figure 3: Organization of the DMD gene and its protein products. ........................................

Figure 4 :

 4 Figure 4: Dystrophin and the dystrophin associated protein complex (DAPC) in muscle tissue. ......................................................................................................................

Figure 5 :

 5 Figure 5: Flowchart for the generation of Dmd EGFP reporter mice. .........................................

Figure 6 :

 6 Figure 6: Strategy for genomic modification of the murine Dmd locus. .................................

Figure 7 :

 7 Figure 7: Subcloning of the 10.943 kb Dmd exon 79 genomic fragment by gap repair. .........

Figure 8 :

 8 Figure 8: Generation of the PL452+mini targeting cassette. ...................................................

Figure 9 :

 9 Figure 9: Generation of the targeting vector. ..........................................................................

Figure 10 :

 10 Figure 10: Functionality test #1: Expression of the Dmd exon 79*-FLAG-EGFP cassette in mammalian cells. .....................................................................................................

Figure 11 :

 11 Figure 11: Functionality test #2: Excision of neo cassette. ......................................................

Figure 12 :

 12 Figure 12: Detection of homologous recombination in ES cells via Southern blot analysis. ..

Figure 13 :

 13 Figure 13: Chimeric mice generated from ES cell clone 6. ......................................................

Figure 14 :

 14 Figure 14: Genotyping PCR and Southern blot analysis for the targeted allele. .....................

Figure 15 :

 15 Figure 15: Genotyping PCR for the targeted dystrophin allele and neo cassette excision in transgenic mice. ......................................................................................................

Figure 16 :

 16 Figure 16: Native EGFP expression in tibialis anterior (TA) muscle sections. ..........................

Figure 17 :

 17 Figure 17: Co-localization of EGFP-and FLAG-tag in tibialis anterior (TA) muscle sections. ..

Figure 18 :

 18 Figure 18: Western blot analysis of dystrophin-EGFP expression in tibialis anterior (TA) muscles. ...................................................................................................................

Figure 19 :

 19 Figure 19: Correct localization of the EGFP-tagged dystrophin at the sarcolemma. ..............

Figure 20 :

 20 Figure 20: EGFP expression in the heart. .................................................................................

Figure 21 :

 21 Figure 21: EGFP expression in smooth muscle. .......................................................................

Figure 22 :

 22 Figure 22: Western blot analysis of dystrophin-EGFP expression in the brain. ......................

Figure 23 :

 23 Figure 23: EGFP expression in brain. .......................................................................................

Figure 24 :

 24 Figure 24: EGFP expression in the retina. ................................................................................

Figure 25 :

 25 Figure 25: Dmd EGFP reporter mice show normal histopathology.............................................

Figure 26 :

 26 Figure 26: Dmd EGFP reporter mice showed normal histopathology. .......................................

Figure 28 :

 28 Figure 28: EGFP expression in satellite cell derived myotubes. ..............................................

Figure 29 :

 29 Figure 29: Targeting of various dystrophin isoforms in Dmd EGFP reporter mice. ..................

13. 3 .

 3 Figure A2: H&E staining of skeletal and heart muscle of adult and aged wildtype (WT) and Dmd EGFP mice. Skeletal muscles quadriceps (QUAD), gastrocnemius (GAS), extensor digitorum longus (EDL), soleus (SOL), diaphragm (DIA) and heart muscle from adult wildtype and transgenic mice show normal morphology with consistency in fiber caliber and no evidence of central nuclei. No further changes are observed in aged mice in the QUAD, GAS, EDL and heart muscle. Scale bar = 20 µm.

Figure A3 :

 A3 Figure A3: Immunohistochemistry of skeletal muscles of wildtype (WT) and Dmd EGFP mice. Normal sarcolemmal expression of dystrophin, C-terminal (Dys2) and rod domain (Dys1) (A), α-,β-Sarcoglycan (B), γ-Sarcoglycan, α-Dystroglycan (C), nNOS (D) can be observed in following skeletal muscles gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), quadriceps (QUAD) and extensor digitorum longus (EDL). Staining of a TA section only with secondary anti-mouse antibodies using the MOM kit shows no background (E). Scale bar = 50 µm. AB: antibody.

Table 1 : Plastic material for tissue culture and molecular biology

 1

	Plastic material	Supplier
	Cell culture dishes (Ø 10 cm)	Becton Dickinson, Heidelberg, Germany
		Sarstedt, Nürnbrecht, Germany
		TPP, Trasadingen, Switzerland
	Cell culture dishes (Ø 35 cm)	TPP, Trasadingen, Switzerland
	Cell culture flasks (175 cm 2 , 75 cm 2 , 25cm 2 )	TPP, Trasadingen, Switzerland
	Cell culture plates-multiwell	TPP, Trasadingen, Switzerland
	(6, 12, 24, 96 well)
	Conical polysterene tubes	Falcon/ Corning, NY, USA
	(15 ml/ 50 ml)
	Coverslips	Menzel, Braunschweig, Germany
	Coverslips, round	R. Langenbrink, Emmendingen, Germany
	Cryovials	Thermo Fisher Scientific, Langenselbold, Germany
	Cuvettes	Sarstedt, Nürnbrecht, Germany
	Disposable hypodermic needle, 22 G x 1 1/4	Braun, Melsungen, Germany
	Film, Fuji Super RX	Röntgen-Bender, Baden-Baden, Germany
	Hybond-XL TM -Transfermembran	GE Healthcare, Munich, Germany
	Insulin syringes	VWR, Darmstadt, Germany
	Pasteur Pipettes	Assistent, Hecht, Sondheim v. d. Rhön, Germany

Table 2 : Instruments and equipment for molecular biological work

 2

	Instruments	Supplier
	ABI PRiSM Genetic analyzer 3500	Thermofisher Scientific, Berlin, Germany
	Analytical balances Kern 770, BP 3100 S	Sartorius Göttingen, Germany
	Autoclave Systec V150	Systec GmbH, Wettenberg, Germany
	Centrifuge 5415C	Eppendorf, Hamburg, Germany
	Centrifuge 5804R	Eppendorf, Hamburg, Germany
	DNA-electrophoresis chambers	BioRad, Munich, Germany LifeTechnologies/Gibco, CA, USA
	Electroporator	BioRad, Munich, Germany
	Electrophoresis unit Ruby SE600	Amersham/Hoeffer, Germany
	Magnetic stirrer	Janke & Kunkel GmbH & Co. KG -IKALabortechnik,
		Staufen, Germany
	Molecular Biology Incubator, Heraus	Heraeus, Hanau, Germany
	Microwave	Sharp, Germany
	NanoDrop ND-1000 Spectrophotometer	Peqlab, Erlangen, Germany
	PH-Meter	Greisinger, Regenstauf, Germany
	Precision balance	Sartorius, MA, USA
	Powersupply	Biometra, Göttingen, Germany BioRad, Munich, Germany
	Rotator	MS-L GmbH, Wiesloch
	Semidry Blot	Biometra, Göttingen, Germany
	Shaker TH25	Edmund Bühler, Tübingen
	Shaking incubator	New Brunswick Edmund Bühler GmbH, Hechingen, Germany
	Spectrophotometer Helios	Thermo Spectronic, Wuppertal, Germany
	Table Centrifuge Ministar	VWR, Darmstadt, Germany

Table 3 : Instruments and equipment for tissue culture work

 3

	Instruments	Supplier
	Biological Safety Cabinet Herasafe	Heraeus, Hanau, Germany
	Cell Culture Incubator, Hera cell, Hera cell150	Thermofisher Scientific, Berlin, Germany
	Centrifuge 5415R	Eppendorf, Hamburg, Germany
	Centrifuge Rotofix32A	Hettich, Tuttlingen, Germany
	Freezing chambers Nalgene® Mr Frosty	Thermo Scientific, Dreieich, Germany
	Microscope Inverted Leica DMI 3000B	Leica Microsystems, Wetzlar, Germany
	Pipetboy acu	Eppendorf, Hamburg, Germany
		Eppendorf, Hamburg, Germany
	Pipettes	Gilson, Bad Camberg, Germany
		Finnpipette, Dreieich, Germany
	Stereomicroscope	Schott, Mainz, Germany
	Vortexer Janke & Kunkel GmbH & Co. KG	IKA Labortechnik, Staufen, Germany
	Vortexer	Heidolph, Schwabach, Germany
	Water bath	GFL, Burgwedel, Germany

Table 4 : Instruments and equipment for histological work

 4

	Instruments	Supplier
	Cryostat, Leica CM 1900	Leica Microsystems, Wetzlar, Germany
	Fluorescence microscope	Leica Microsystems, Wetzlar, Germany
	Fluorescence light source	Leica Microsystems, Wetzlar, Germany
	Fluorescence light source X-Cite 120	Excelitas Technologies, Wiesbaden, Germany
	Rolera MGI camera	Qimaging, BC, Canada
	SPOT RT3 camera	Spotimaging, MI, USA
	Surgical scissors	neoLab Migge Laborbedarf-Vertriebs, Heidelberg,
		Germany
	Tweezers	GFL, Burgwedel, Germany

Table 5 : Chemicals

 5

	Chemicals	Supplier
	Acetone	Merck, Darmstadt, Germany
	Acrylamide/Bisacrylamide 40%, 29:1	BioRad, Munich, Germany
	Agarose MP	Roche, Grenzach-Wyhen, Germany
	Albumine bovine fraction V	Serva, Heidelberg, Germany
	Ampicillin sodium salt	Carl-Roth, Karlsruhe, Germany
	Arabinose	AppliChem, Darmstadt, Germany
	Bisodium Hydrogenphosphate-Dihydrate	Carl-Roth, Karlsruhe, Germany
	Boric acid	Merck, Darmstadt, Germany

Kit systems, markers, enzymes and nucleotides

Table 6

 6

	: Kits
	Kit	Supplier
	BigDye® Terminator v3.1 Cycle Sequencing Kit	Applied Biosystems, Weiterstadt, Germany
	HiSpeed Plasmid Midi Kit	Qiagen, Hilden, Germany
	Lipofectamin, LTX	Thermo Fisher Scientific, Wuppertal, Germany
	MOM Kit	Vector Laboratories, Enzo, Lörrach, Germany
	pGEMT-easy Vector System 1	Promega, Mannheim, Germany
	QIAprep® Spin Miniprep Kit	Qiagen, Hilden, Germany
	QIAquick® Gel Extraction Kit	Qiagen, Hilden, Germany
	QIAquick® PCR Purification Kit	Qiagen, Hilden, Germany
	Quickligase Kit	New England Biolabs, Frankfurt, Germany
	VenorGEM mycoplasma diagnosis test	Biochrom, Berlin, Germany

Table 7 : Standard markers

 7

	Markers	Supplier
	100 bp Standard marker	New England Biolabs, Frankfurt, Germany
	1 kb Standard marker	New England Biolabs, Frankfurt, Germany
	Precision Plus Protein ™ All Blue Prestained Protein	BioRad, Munich, Germany
	Standards
	HiMark™ Pre-stained Protein Standard	Thermo Fisher Scientific, Wuppertal, Germany
	Table 8: Enzymes
	Enzymes	Supplier
	Calf intestine Phosphatase	New England Biolabs, Frankfurt, Germany
	DNaseI	Invitrogen, Karlsruhe, Germany
	GoTaq TM DNA-Polymerase	Promega, Mannheim, Germany
	Proteinase K	AppliChem, Darmstadt, Germany
	Phusion TM DNA-Polymerase	Finnzymes, Espoo, Finnland
	Restriction Enzymes	New England Biolabs, Frankfurt, Germany
	Quickligase	Promega, Mannheim, Germany
	Table 9: Nucleotides
	Markers	Supplier
	dNTPs (je 100 mM)	Promega, Mannheim, Germany

Table 12 : Cell lines, primary murine cells and media used for culturing Cell line/Primary cells

 12

	Description/medium

5.10. Animal experiments 5.10.1. Mouse strains and animal husbandry conditions Animals were bred and kept under specific pathogen-free (SPF)-like conditions in the animal facilities of the Charité Cross Over building, as well as in the central animal facility FEM of the Charité Universitätsmedizin Berlin. Animals for organ preparation were sacrificed by cervical disloction. The experiments were performed in compliance with the local animal experimentation guidelines. Animal experiments were approved by the local authorities (LaGeSo Berlin, T 0222/13).

Table 13 : Mouse strains Name/Reference Internal name

 13

	Supplier

  It is suitable for modification of murine genomes and for the cloning of long fragments. This method exploits a phage-based recombination system in E. coli which forces the introduction of a linear DNA of interest sequence into a plasmid provided there is enough homology at the 5' and 3' end of the DNA of interest. The proteins responsible for homologous recombination are produced by a replication defective λ Phage integrated into the bacterial genome. The red genes exo, bet and gam regulate the homologous recombination allowing insertion of double stranded DNA fragments into plasmids, BACs (bacterial artificial chromosomes) or PACs (P1-derived artificial chromosomes). The genes are under the control of the PL promoter und a temperature sensitive repressor cI857. At low temperature (30-34°C) the repressor is active, but raising the temperature to 42°C leads to deactivation of the repressor and expression of the red genes.

. Recombineering is a molecular genetic technique based on genetic engineering by in vivo DNA manipulation in Escherichia coli (E. coli) via homologous recombination. This system allows the construction of DNA molecules with precise junctions and is independent of the location of appropriate restriction sites, which overcomes the limitations of conventional cloning strategies.

Table 1 :

 1 Plastic material for tissue culture and molecular biology .........................................

Table 2 :

 2 Instruments and equipment for molecular biological work ......................................

Table 3 :

 3 Instruments and equipment for tissue culture work .................................................

Table 4 :

 4 Instruments and equipment for histological work ....................................................

Table 5 :

 5 Chemicals ...................................................................................................................

Table 6 :

 6 Kits ..............................................................................................................................

Table 7 :

 7 Standard markers .......................................................................................................

Table 8 :

 8 Enzymes ......................................................................................................................

Table 9 :

 9 Nucleotides ................................................................................................................

Table 10 :

 10 Plasmids ...................................................................................................................

Table 11 :

 11 Bacteria ....................................................................................................................

Table 12 :

 12 Cell lines, primary murine cells and media used for culturing ................................

Table 13 :

 13 Mouse strains ...........................................................................................................

Table 14 :

 14 Software ...................................................................................................................

13.2. Plasmid maps Figure A1: Plasmidmaps

IHC of skeletal muscle sections

Incubation of the blots with anti-GFP antibody revealed a strong band running at approximately 100 kDa in the transgenic samples which would correspond to an EGFP-tagged Dp71 isoform. No bands were detected in either of the samples using the rod-domain specific anti-Dys1 antibody. Furthermore, we analyzed brain sections for dystrophin-EGFP expression. In Figure 23 natural EGFP expression in the cerebellum of Dmd EGFP mice is depicted. The EGFP signal showed colocalization with the anti-dystrophin signal. Moreover, brain blood vessels expressed EGFP as demonstrated by co-labeling the vessels with anti-CD31 and partial co-localization of the respective fluorescence signals (Figure 23A). In addition, we detected expression of dystrophin in glial cells localized at the glial-vascular interface in the cerebellum as well as in the hippocampus, shown by a partial co-localization of the EGFP signal with the immune signal from an antibody against the glial fibrillary acidic protein (GFAP).

In the retina of Dmd EGFP reporter mice, EGFP expression was observed at the outer plexiform layer (OPL), the inner limiting membrane (ILM) and sporadically at the inner nuclear layer (INL) (Figure 24A). A strong punctate EGFP signal co-localized with dystrophin immunofluorescence and was detectable in the OPL of the retina (Figure 24B). An exact colocalization of the EGFP signal was detected when using a C-terminal specific anti-dystrophin antibody (Dys2). Using the rod domain specific MANDYS19 antibody, not all EGFP positive structures were stained confirming that we have obviously targeted different isoforms. EGFP expression was also observed in at the inner nuclear layer. In addition, in retinal blood vessels of reporter mice the EGFP signal also co-localized with anti-CD31 signals. Dystrophin-EGFP at the blood vessels was not identified with the MANDYS19 antibody. At the ILM the principal glial cells of the retina -the Müller cells -were stained with the anti-GFAP antibody. Colocalization of the GFAP immunosignal in this region with EGFP expression was observed only at certain regions of the glial cells, most probably the glial endfeet (Figure 24C).

The EGFP expression patterns in brain and retina thus verify the successful tagging of different dystrophin isoforms in the Dmd EGFP reporter mice.

Histological analysis of the Dmd EGFP reporter mice

The C-terminal domain of dystrophin plays an important role in the binding and assembly of the DAPC, which has a crucial role for muscle function and sarcolemmal stability. Addition of a 27.9 kDa EGFP-tag to the C-terminus of dystrophin might interfere with proper protein

*

Dystrophin exerts its most important function through assembly of the DAPC complex, which is responsible for its mechanical as well as its signaling functions. The addition of a 27.9 kDa fluorescent protein might change the 3D-folding of dystrophin or mask its binding sites thereby disrupting important interactions. Loss of expression of DAPC components generally destabilizes the sarcolemma and triggers dystrophic changes [START_REF] Kanagawa | The genetic and molecular basis of muscular dystrophy: roles of cell-matrix linkage in the pathogenesis[END_REF].

The protein structure of GFP comprises 11 β-sheets with an internal α-helix and short helical segments on the ends of the cylinder, which constitute a protein folding structure named βcan. The fluorophores are protected inside the cylinders. Residues on the outside are predominantly polar. In the case of standard GFP, the dimerization domain consists of a small hydrophobic patch and numerous hydrophilic contacts [START_REF] Yang | The molecular structure of green fluorescent protein[END_REF]. In the mutant EGFP-p.L221K used by us, this hydrophobic interaction is disrupted. For the C-terminus of dystrophin, which is expressed from exons 78-79, the predicted folding pattern is a β-sheet with few hydrophobic residues (≈16%) at the surface and a global positive charge.

For the splice variant that encodes the embryonic Dp71 (Dp71f) isoform, an alternative splicing event excludes exon 78 and generates a modified C-terminus [START_REF] Austin | Cloning and characterization of alternatively spliced isoforms of Dp71[END_REF].

The new ORF has a stop codon, which is located downstream in the 3'UTR and produces a dystrophin with a 31 amino acid tail instead of the shorter 13 amino acid tail if exon 78 would be included. With the alternative C-terminus, the newly created amphipathic α-helix contains a higher percentage of hydrophobic residues and a global negative charge, which has been suggested to confer different biophysical and functional properties [START_REF] Rau | Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy[END_REF]. In order to exclude steric incompatibilities of the EGFP tag, which constitutes a significant addition to an important functional domain of the protein and may thus have unwanted effects on protein folding, function, or targeting we analyzed the morphology of the muscle. H&E staining of cross sections from different skeletal as well as from heart muscle did not show any differences between the adult reporter mice and their wildtype littermates (Figure 25, Appendix 13.3, Figure A2). Moreover, no dystrophic changes were detected in aged Dmd EGFP (10 months old) mice, which were well beyond the age, when dystrophic changes are commonly seen in mdx mice. For comparison, we analyzed aged-matched dystrophic mdx mice, which showed strong muscle degeneration, fibrosis, immune cell infiltration, and central nucleation -all hallmarks of an advanced dystrophic process.

periphery. The distribution of the signal was punctuate and this is in line with previous studies using dystrophin antibodies to depict dystrophin in the duodenal wall [START_REF] Byers | The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle[END_REF]. This distribution is due to the exclusion of dystrophin from the adherens junctions between smooth muscles. This was demonstrated in smooth muscle of guinea pigs, where dystrophin and vinculin, a protein expressed in adherens junctions, had a mutually exclusive location [START_REF] North | Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle[END_REF][START_REF] Porter | Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle[END_REF].

In the colon of Dmd EGFP reporter mice, we were also able to demonstrate co-localization between the anti-dystrophin immune signal and EGFP fluorescence (Figure 21B). For the labeling dystrophin in the colon we used a rabbit polyclonal C-terminal anti-dystrophin antibody in order to evade unspecific staining by the secondary anti-mouse IgG antibody since we expected a higher presence of immune cells and of immune globulin deposits in the gut of the mouse.

Non-muscle dystrophin isoforms are successfully tagged in the brain and retina of

Dmd EGFP reporter mice

The highest variety of dystrophin isoforms can be found in the brain and the short Dp71 is the predominant one. Brain isoforms commonly include the domains involved in β-dystroglycan and syntrophin binding, but lack the actin binding domains [START_REF] Culligan | Diversity of the Brain Dystrophin-Glycoprotein Complex[END_REF].

Full-length dystrophin in the brain is expressed almost exclusively in neurons of the cerebellum, cortex and hippocampus [START_REF] Lidov | Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons[END_REF]. Different studies have reported that dystrophin and DAPC-like complexes are involved in modulating synapse function at a subset of GABAergic synapses in the hippocampus and cerebellum of mice. An association with clusters of inhibitory GABAA receptors has been reported [START_REF] Brünig | GABAergic terminals are required for postsynaptic clustering of dystrophin but not of GABA(A) receptors and gephyrin[END_REF][START_REF] Kneussel | Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice[END_REF]. As already mentioned in the introduction, a functional proof of the interaction between dystrophin and the GABAA receptor has not yet been presented. However, it is important to understand the role of dystrophin and its DAPC at the inhibitory synapses as it might explain some of the behavioral and electrophysiological findings in dystrophin deficient mdx mice [START_REF] Anderson | Altered inhibitory input to Purkinje cells of dystrophin-deficient mice[END_REF][START_REF] Graciotti | GABAergic miniature spontaneous activity is increased in the CA1 hippocampal region of dystrophic mdx mice[END_REF][START_REF] Kueh | GABAA RECEPTOR EXPRESSION AND INHIBITORY POST-SYNAPTIC CURRENTS IN CEREBELLAR PURKINJE CELLS IN DYSTROPHIN-DEFICIENT mdx MICE[END_REF].

Other authors have proposed the expression of dystrophin at the postsynaptic density which mainly contains excitatory neurotransmitter receptors [START_REF] Kim | Detection of dystrophin in the postsynaptic density of rat brain and deficiency in a mouse model of Duchenne muscular dystrophy[END_REF]. These contradictory 13. APPENDIX

List of primers