German Aerospace Center ANALYSIS OF NUCLEAR MORPHOLOGY OF DESMIN KNOCK-OUT MYOBLASTS Bachelor thesis Submitted by Elena Anna-Maria Honscheid Cologne, July 2020 This study was conducted from May to July 2020 at the Institute of Aerospace-Medicin, Ger- man Aerospace Center, Cologne, in the work group “Molecular Muscle and Bone Research” under the supervision of Prof. Dr. med. Christoph S. Clemen. 1st supervisor: Prof. Dr. rer. nat. Jan Riemer Institute of Biochemistry University of Cologne 2nd supervisor: Prof. Dr. rer. nat. Ines Neundorf Institute of Biochemistry University of Cologne Eidesstattliche Erklärung Hiermit versichere ich an Eides statt, dass ich die vorliegende Arbeit selbstständig und ohne die Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Alle Stellen, die wörtlich oder sinngemäß aus veröffentlichten und nicht veröffentlichten Schriften entnommen wurden, sind als solche kenntlich gemacht. Die Arbeit ist in gleicher oder ähnlicher Form oder auszugsweise im Rahmen einer anderen Prüfung noch nicht vorgelegt worden. Ich versichere, dass die eingereichte elektronische Fassung der eingereichten Druckfassung vollständig entspricht. Ich bin mir bewusst, dass die falsche Abgabe der Versicherung an Eides statt die Rechtsfolgen des § 63 Absatz 5 HG Anwendung finden. ________________________ Ort, Datum ___________________________________ Unterschrift TABLE OF CONTENT ABSTRACT ...................................................................................................................................................... 1 1 INTRODUCTION ................................................................................................................................ 4 1.1 CYTOSKELETON ................................................................................................................................... 4 1.2 INTERMEDIATE FILAMENTS ................................................................................................................. 4 1.3 DESMIN ............................................................................................................................................... 5 1.4 DESMINOPATHIES AND MYOFIBRILLAR MYOPATHIES .......................................................................... 5 1.4.1 DESMIN KNOCK-OUT PATIENTS ...................................................................................................... 6 1.5 DESMIN KNOCK-OUT MICE .................................................................................................................. 6 1.6 DESMIN KNOCK-DOWN IN CARDIOMYOCYTES .................................................................................... 6 1.7 MYOBLASTS AND MYOGENESIS ............................................................................................................ 7 1.8 NUCLEAR MORPHOLOGY ..................................................................................................................... 8 1.9 LINC COMPLEX ..................................................................................................................................... 8 1.10 HYPOXIA .............................................................................................................................................. 9 1.11 AIM OF THIS STUDY ............................................................................................................................. 9 2 MATERIALS AND METHODS ............................................................................................................ 10 2.1 MATERIALS ........................................................................................................................................ 10 2.1.1 BUFFERS ........................................................................................................................................ 10 2.1.2 ENZYMES ...................................................................................................................................... 10 2.1.3 INHIBITORS ................................................................................................................................... 10 2.1.4 MAMMALIAN CELL LINES ............................................................................................................. 10 2.1.5 CELL CULTURE MEDIUM ............................................................................................................... 11 2.1.6 PCR-PRIMER .................................................................................................................................. 11 2.1.7 ANTIBODIES .................................................................................................................................. 11 2.1.8 MISCELLANEOUS ........................................................................................................................... 12 2.2 METHODS .......................................................................................................................................... 12 2.2.1 COATING OF CELL CULTURE DISHES ............................................................................................. 12 2.2.2 GENERATION OF MURINE MYOBLAST CELL LINES ........................................................................ 13 2.2.3 REVITALIZATION OF MYOBLASTS .................................................................................................. 13 2.2.4 CULTIVATION OF MYOBLASTS ...................................................................................................... 13 2.2.5 DIFFERENTIATION OF MYOBLASTS ............................................................................................... 14 2.2.6 OXYGEN CONDITIONS ................................................................................................................... 15 2.2.7 ISOLATION OF GENOMIC DNA ...................................................................................................... 15 2.2.8 POLYMERASE CHAIN REACTION ................................................................................................... 15 2.2.9 DNA AGAROSE GEL ELECTROPHORESIS ........................................................................................ 16 2.2.10 CELL SAMPLES FOR IMMUNFLUORESCENCE MICROSCOPY .......................................................... 16 2.2.11 IMMUNOFLUORESCENCE STAINING ............................................................................................. 17 2.2.12 MICROSCOPY ................................................................................................................................ 19 2.2.13 TIME LAPSE MOVIES WITH JuLITM Br ............................................................................................. 20 2.2.14 DETERMINATION OF NUCLEAR SHAPE ......................................................................................... 20 2.2.15 MEASURING THE NUMBER OF !H2AX SPOTS ............................................................................... 20 2.2.16 MUSCLE TISSUE SLICES ................................................................................................................. 20 2.2.17 STATISTICAL ANALYSES ................................................................................................................. 21 3 RESULTS ......................................................................................................................................... 22 3.1 GENOTYPING ..................................................................................................................................... 22 3.2 MYOBLAST GROWTH ......................................................................................................................... 22 3.3 MYOBLAST DIFFERENTIATION ........................................................................................................... 23 3.4 IMMUNOFLUORESCENCE STAININGS ................................................................................................ 24 3.4.1 NUCLEAR MORPHOLOGY .............................................................................................................. 25 3.4.2 DNA DAMAGE ............................................................................................................................... 28 4 DISCUSSION ................................................................................................................................... 30 4.1 MYOBLAST GROWTH AND DIFFERENTIATION ..................................................................................... 30 4.2 NUCLEAR MORPHOLOGY ................................................................................................................... 30 4.3 NUCELAR SIZE .................................................................................................................................... 31 4.4 DNA DAMAGE .................................................................................................................................... 32 4.5 ERROR ANALYSIS ................................................................................................................................ 33 5 CONCLUSION AND OUTLOOK ......................................................................................................... 33 6 REFERENCES ................................................................................................................................... 34 7 APPENDIX ......................................................................................................................................