CHARGE syndrome: candidate genes and pathogenesis Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program Genes and Development of the Georg-August University School of Science (GAUSS) submitted by Yvonne Schulz from Hamburg, Germany Göttingen 2014 Thesis Committee Prof. Dr. med. Dr. h.c. Wolfgang Engel Department of Human Genetics, Georg-August-University, Göttingen Prof. Dr. Andreas Wodarz Department of Microscopical Anatomy and Molecular Cell Biology, University Köln Medical Center, Köln Prof. Dr. Ahmed Mansouri Department of Molecular Cell Biology/Molecular Cell Differentiation, Max Planck Institute for biophysical Chemistry, Göttingen, Members of the Examination Board Referee: Prof. Dr. med. Dr. h.c. Wolfgang Engel Department of Human Genetics, Georg-August-University, Göttingen 2nd Referee: Prof. Dr. Andreas Wodarz Department of Microscopical Anatomy and Molecular Cell Biology, University Köln Medical Center, Köln Further members of the Examination Board Prof. Dr. Sigrid Hoyer-Fender Department of Developmental Biology GZMB, Johann-Friedrich-Blumenbach- Institute for Zoology and Anthropology, Georg-August-University Göttingen Prof. Dr. Ernst Wimmer Department of Developmental Biology GZMB, Johann-Friedrich-Blumenbach- Institute for Zoology and Anthropology, Georg-August-University Göttingen Prof. Dr. Steven Johnsen Clinic for General, Visceral and Pediatric Surgery, University Medical Center, Göttingen Date of the oral examination: Herewith I declare, that I prepared the Dissertation "CHARGE syndrome: candidate genes and pathogenesis" on my own and with no other sources and aids than quoted. ______________________ Göttingen, August 20th, 2014 Yvonne Schulz Dedicated to my family Acknowledgements I would like to express my sincere gratitude to my doctor father Prof. Dr. med. Dr. h.c. Wolfgang Engel for the opportunity to compile my PhD study in the Institute of Human Genetics. I am overgrateful for the valuable scientific discussions and his encouragement during my PhD study. I would like to thank the members of my thesis committee Prof. Dr. Andreas Wodarz and Prof. Dr. Ahmed Mansouri for their readiness to invest their time and for the valuable input during our meetings. I would like to express my deep gratitude to my supervisor PD Dr. Silke Pauli for her unquestioning support, patience and trust and excellent guidance during the entire process of my PhD project. I am thankful for the facilitation to work in such interesting field of research. Moreover, I thank Prof. Dr. Sigrid Hoyer-Fender, Prof. Dr. Ernst Wimmer and Prof. Dr. Steven Johnsen, who kindly agreed to evaluate my dissertation and participate in examination. I sincerely thank our collaborators, especially Prof. Dr. Annette Borchers and Dr. Peter Wehner. I would particularly like to thank the staff of the Goettingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences (GGNB) and my doctoral program “Genes and Development” for the excellent organisation and financial support by the grant of the bridging fund as well as the Deutsche Forschungsgemeinschaft (DFG) for financing my PhD study. Special thanks go to Johanna Mänz, who always supported my work with great assistance. Further, I would like to thank the staff of animal keeper for excellent animal care. I would like to thank my colleagues Johanna Mänz, Luisa Freese, Jessica Nolte, Nadine Mellies, Stefanie Heumüller, Tserendulam Batsukh, Krzysztof Wieczerzak and all other members of the Human Genetic Institute for their advice and support, friendship and great working atmosphere. Herewith, I would like to express my heartfelt gratitude to my family, especially to my parents who always supported me with their trust and love. I wish my dad could share this special moment of my life with me... Thank you to Benjamin Brauer and all my friends who accompanied me through this part of my life journey. Because of you this time became an unforgettable one and will always have a special place in my heart! Thank you so much! Table of content Table of content...........................................................................................................I List of figures............................................................................................................VI List of tables...........................................................................................................VIII Abbreviations.............................................................................................................X Nomenclature..........................................................................................................XX Summary................................................................................................................XXI 1 Introduction ................................................................................................. 1 1.1 The chromodomain helicase DNA-binding protein 7 ................................... 1 1.2 CHD7 exists in large multi-subunit complexes ............................................. 1 1.3 CHARGE syndrome ...................................................................................... 3 1.4 Neural crest cells - the explorer of the vertebral embryo .............................. 4 1.5 NCC development ......................................................................................... 6 1.6 NCC guidance and signalling factors ............................................................ 7 1.7 Xenopus laevis as a model organism for studying NCC development .......... 9 1.8 The mouse as a model organism and its advantages ................................... 10 1.9 Aim of the work .......................................................................................... 11 2 Materials and methods.............................................................................. 13 2.1 Materials ...................................................................................................... 13 2.1.1 Instruments ........................................................................................... 13 2.1.2 Consumable materials .......................................................................... 15 2.1.3 Kits ....................................................................................................... 17 2.1.4 Ready to use buffers and mediums ...................................................... 17 2.1.5 Chemicals ............................................................................................. 18 2.1.6 Buffers and solutions ........................................................................... 22 2.1.7 Media and plates .................................................................................. 28 2.1.8 Sterilisation .......................................................................................... 30 I 2.1.9 Antibiotics ............................................................................................ 30 2.1.10 Antibodies ............................................................................................ 30 2.1.11 Morpholinos ......................................................................................... 31 2.1.12 Oligonucleotides .................................................................................. 32 2.1.13 DNA marker ......................................................................................... 39 2.1.14 Protein marker ...................................................................................... 39 2.1.15 Vectors ................................................................................................. 39 2.1.16 Enzymes ............................................................................................... 40 2.1.17 Polymerases (Kits) ............................................................................... 40 2.1.18 Restriction enzymes and Buffers ......................................................... 40 2.1.19 Bacterial strains .................................................................................... 40 2.1.20 Cell line ................................................................................................ 41 2.1.21 Yeast strain ........................................................................................... 41 2.1.22 Model organisms .................................................................................. 41 2.1.23 Software used ....................................................................................... 41 2.1.24 Internet platforms used ......................................................................... 41 2.2 Methods ....................................................................................................... 43 2.2.1 Isolation of nucleic acids ...................................................................... 43 2.2.1.1 Isolation of genomic DNA for genotyping of mice ...................... 43 2.2.1.2 Isolation of plasmid DNA from bacteria ...................................... 43 2.2.1.3 RNA isolation from mouse embryos ............................................ 45 2.2.2 Determination of nucleic acid concentration ....................................... 46 2.2.3 Reverse transcription ............................................................................ 46 2.2.4 Cloning ................................................................................................. 46 2.2.4.1 Restriction digestion of plasmid