Generation and Characterization of a DmdEGFP Reporter Mouse as a Tool to Investigate Dystrophin Expression Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin and in cotutelle to the Ecole Doctorale 515 "Complexité du vivant" of Université Pierre et Marie Curie Paris by Mina Petkova from Sofia, Bulgaria 2015 The research presented in this thesis was conducted from October 2011 until December 2015 in the followng laboratories: Department of neuropediatrics and NeuroCure, Charité Cross Over, Charité Universitätmedizin, Berlin, Germany. Biothérapies des maladies neuromusculaires, UFR des sciences de la santé Simone Veil, Université de Versailles Saint-Quentin-en-Yvelines, France. THESIS COMMITTEE: Thesis supervisor and reviewer Freie Universität Berlin: Prof. Dr. Markus Schuelke Thesis supervisor Université Pierre et Marie Curie Paris: Prof. Dr. Helge Amthor Representative Université Pierre et Marie Curie Paris: Dr. Gillian Butler-Browne Reviewer Freie Universität Berlin: Prof. Dr. Sigmar Stricker External reviewer: Prof. Dr. Volker Straub External reviewer: Prof. Dr. Ketan Patel Postdoctoral fellow Freie Universität Berlin: Dr. Annemarie Hofmann Date of defense: 05.02.2016 STATUTORY DECLARATION I hereby declare that I wrote the presented dissertation “Generation and characterization of a DmdEGFP 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. Berlin, 6.01.2016 _______________ (Signature) ACKNOWLEDGMENTS I would like 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. 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) TABLE OF CONTENTS TABLE OF CONTENTS 1. Summary ................................................................................................................... 1 2. Zusammenfassung ..................................................................................................... 2 3. Résumé ..................................................................................................................... 4 4. Introduction .............................................................................................................. 6 4.1. Pathology of Duchenne muscular dystrophy ....................................................... 6 4.2. The mdx mouse: a naturally occurring animal model for DMD ............................ 8 4.3. The structure and function of the muscle ........................................................... 9 4.3.1. Skeletal muscle ....................................................................................................... 9 4.3.2. Cardiac and smooth muscle ................................................................................. 11 4.3.3. Myogenesis ........................................................................................................... 12 4.3.4. The satellite cell and skeletal muscle regeneration ............................................. 12 4.4. Dystrophin ....................................................................................................... 14 4.4.1. The DMD gene ...................................................................................................... 14 4.4.2. The dystrophin protein in the skeletal muscle ..................................................... 16 4.4.3. Dystrophin associated protein complex (DAPC) .................................................. 18 4.4.4. Utrophin: a dystrophin homolog .......................................................................... 21 4.4.5. Dystrophin in cardiac and smooth muscles ......................................................... 22 4.4.6. Dystrophin in non-muscle tissues ........................................................................ 24 4.4.7. The role of dystrophin in satellite cells ................................................................ 28 4.5. Mutations of the DMD gene ............................................................................. 29 4.6. Revertant fibers in DMD patients and in mdx mice ........................................... 30 4.7. Therapies for DMD ........................................................................................... 31 4.8. Aims of the study ............................................................................................. 33 5. Materials ................................................................................................................. 36 5.1. Plastic materials ............................................................................................... 36 5.2. Instruments, equipment................................................................................... 37 5.3. Chemicals ........................................................................................................ 38 5.4. Kit systems, markers, enzymes and nucleotides................................................ 40 5.5. Plasmids .......................................................................................................... 41 5.6. Bacteria ........................................................................................................... 41 5.7. Antibodies ....................................................................................................... 41 5.8. Oligonucleotide primers for PCR....................................................................... 41 5.9. Cell lines .......................................................................................................... 42 5.10. Animal experiments ......................................................................................... 42 i 5.11. Software .......................................................................................................... 42 6. Methods ................................................................................................................. 44 6.1. Methods for the generation of a transgenic Dystrophin-EGFP reporter mouse .. 44 6.2. Generation of the