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Kristinkesslerdissertation.Pdf Identifikation und Charakterisierung pathogenetischer Zusammenhänge des Kurzrippen-Polydaktylie Syndroms Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Kristin Keßler Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 28.08.2019 Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer Gutachter: Prof. Dr. med. André Reis PD Dr. Dieter Engelkamp Die Neigung der Menschen, kleine Dinge für wichtig zu halten, hat sehr viel Großes hervorgebracht. Georg Christoph Lichtenberg (1742 - 1799) © KK 08.11.2012 Quilling-Technik: Fibroblast mit primärem Cilium Grün: Cytoplasma, rot: primäres Cilium, blau: Zellkern I Inhaltsverzeichnis Inhaltsverzeic hnis Ihn Inhal tsverzeichnis Inhaltsverzeichnis ......................................................................................................... II 1. Zusammenfassung ............................................................................................................. 1 2. Summary ............................................................................................................................ 3 3. Einleitung ........................................................................................................................... 5 3.1. Aufbau und Funktion von Cilien .............................................................................. 5 3.2. Ciliogenese und intraflagellarer Transport ............................................................. 8 3.3. Ciliopathien ............................................................................................................... 10 3.4. Kurzrippen-Polydaktylie Syndrome ...................................................................... 13 3.5. Zielsetzung dieser Arbeit ......................................................................................... 16 4. Methoden ......................................................................................................................... 17 4.1. Patientenkollektiv ..................................................................................................... 17 4.2. Isolierung von Nukleinsäuren ................................................................................. 19 4.2.1. Isolierung von genomischer DNA aus humanem Blut ..................................... 19 4.2.2. Isolierung von genomischer DNA aus Zellen .................................................. 19 4.2.3. Isolierung von RNA aus humanem Blut ........................................................... 19 4.2.4. Isolierung von RNA aus Zellen ........................................................................ 19 4.2.5. Isolierung von Plasmid-DNA ........................................................................... 20 4.2.6. Qualitätskontrolle für isolierte Nukleinsäuren.................................................. 20 4.3. Gelelektrophorese .................................................................................................... 20 4.3.1. Gelextraktion aus Agarosegelen ....................................................................... 20 4.4. Amplifikation genomischer DNA (Whole Genome Amplification) ....................... 20 4.5. Polymerase-Kettenreaktion (PCR) ......................................................................... 21 4.5.1. Standard-PCR ................................................................................................... 21 4.5.2. GC-rich-PCR .................................................................................................... 22 4.5.3. Hefe-PCR .......................................................................................................... 23 4.6. Reverse Transkriptase PCR (RT-PCR) ................................................................... 24 4.7. Quantitative real time PCR (qPCR) ....................................................................... 24 4.7.1. Durchführung der qPCR ................................................................................... 25 4.7.2. Auswertung qPCR ............................................................................................ 25 4.8. Sequenzierung nach Sanger .................................................................................... 26 4.8.1. PCR Aufreinigung ............................................................................................ 26 4.8.2. Sequenzierreaktion ........................................................................................... 26 4.8.3. Sequenzieraufreinigung .................................................................................... 27 II Inhaltsverzeichnis 4.8.4. Analyse der Sequenzen ..................................................................................... 27 4.9. Multiplex Ligation-dependent Probe Amplification (MLPA) ................................. 28 4.9.1. MLPA Sonden-Design ..................................................................................... 28 4.9.2. Durchführung der MLPA ................................................................................. 29 4.9.3. MLPA Auswertung ........................................................................................... 29 4.10. Klonierung von Plasmiden ...................................................................................... 29 4.10.1. pCR8/GW/TOPO TA Klonierung ..................................................................... 29 4.10.2. LR Clonase Reaktion (Gateway System) .......................................................... 30 4.10.3. Transformation von Plasmiden in chemisch kompetente Bakterien ................ 30 4.10.4. Selektion positiver Klone ................................................................................. 31 4.10.5. Herstellung von Bakterien-Kryostocks............................................................. 31 4.11. Zellkultur .................................................................................................................. 31 4.11.1. Auftauen von Zelllinien .................................................................................... 31 4.11.2. Kultur von adhärent wachsenden Zellen .......................................................... 31 4.11.3. Splitten von Zellkulturen .................................................................................. 32 4.11.4. Einfrieren von Zellkulturen .............................................................................. 32 4.11.5. Transfektion von adhärenten Zelllinien mit siRNA .......................................... 33 4.11.6. Transfektion von adhärenten Zelllinien mit Plasmiden .................................... 33 4.12. Lichtmikroskopie ..................................................................................................... 34 4.12.1. Fixierung und indirekte Immunfluoreszenzmarkierung von Zellen ................. 34 4.12.2. Dreifachfärbung von Zellen mittels Zenon Labeling Technologie................... 35 4.12.3. Fluoreszenzmikroskopische Analyse................................................................ 35 4.13. CRISPR/Cas9 ........................................................................................................... 36 4.13.1. CRISPR/Cas9 Vektordesign und Transfektion in HEK293T ........................... 37 4.13.2. Fluorescence activated cell sorting (FACS) und Zellernte .............................. 38 4.13.3. T7E1 Mismatch Assay....................................................................................... 38 4.13.4. Identifizierung von Mutationen in CRISPR-Zellklonen................................... 39 4.13.5. Überblick über den Ablauf zur Generierung von CRISPR-Zellklonen ............ 40 4.14. Ko-Immunopräzipitation ........................................................................................ 40 4.15. Western Blot Analyse ............................................................................................... 41 4.15.1. Herstellung Proteinlysat.................................................................................... 41 4.15.2. Konzentrationsbestimmung von Proteinen ....................................................... 42 4.15.3. SDS-Polyacrylamid-Gelelektrophorese ............................................................ 42 4.15.4. Western Blot Transfer und Analyse ................................................................. 43 III Inhaltsverzeichnis 4.16. Herstellung eines polyklonalen Antikörpers gegen NEK1 ................................... 44 4.16.1. Auswahl des Epitopbereichs und Klonierung in pGEX-4T3 ............................ 44 4.16.2. Expression rekombinantes Protein und Aufreinigung im GST-System ........... 45 4.16.3. Testung der Präimmunseren und Immunisierung von Kaninchen ................... 46 4.16.4. Testung der Seren und Aufreinigung des Antikörpers via Hi-Trap Säulen ..... 47 4.17. Zusammenstellung einer Ciliengenliste.................................................................. 48 4.18. Ranglistenerstellung von Kandidatengenen mit ENDEAVOUR ......................... 49 4.19. Hefe-2-Hybrid-System (Y2H) ................................................................................. 50 4.19.1. Die Hefestämme AH109 und Y187 .................................................................. 51 4.19.2. Kultivierung von Saccharomyces cerevisiae .................................................... 52 4.19.3. Herstellung
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