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Untersuchung Von Transkriptionsunterschieden in Retina Und Gehirn Des Ccdc66-Defizienten Mausmodells

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Untersuchung Von Transkriptionsunterschieden in Retina Und Gehirn Des Ccdc66-Defizienten Mausmodells Aus der Abteilung für Humangenetik an der Medizinischen Fakultät der Ruhr-Universität Bochum Leiter: Prof. Dr. med. Jörg T. Epplen Untersuchung von Transkriptionsunterschieden in Retina und Gehirn des Ccdc66-defizienten Mausmodells Inaugural-Dissertation zur Erlangung des Doktorgrades der Medizin einer Hohen Medizinischen Fakultät der Ruhr-Universität Bochum vorgelegt von Elke Porrmann-Kelterbaum aus Gelsenkirchen 2017 Dekan: Prof. Dr. med. Albrecht Bufe Referent: Prof. Dr. med. Jörg T. Epplen Korreferent: Prof. Dr. rer. nat. Bernd Eiben Tag der mündlichen Prüfung: 07.06.2018 Meinen Kindern Meike, Sarah, Kristina und Tim Inhaltsverzeichnis 1 Einleitung .........................................................................................................9 1.1 Retinitis pigmentosa .................................................................................................... 9 1.2 Die Retina der Wirbeltiere ......................................................................................... 11 1.2.1 Aufbau der Retina ............................................................................................... 11 1.2.2 Photorezeptorzellen ............................................................................................ 14 1.2.3 Retinale Gliazellen .............................................................................................. 15 1.3 Die Retina der Maus als Modellsystem für retinale Erkrankungen ........................... 17 1.3.1 Histologische Merkmale ...................................................................................... 17 1.3.2 Prä- und postnatale Entwicklung der Retina ....................................................... 18 1.4 Das Ccdc66-/--Mausmodell für retinale Degeneration ............................................... 20 1.4.1 Die Ccdc66-defiziente Maus ............................................................................... 20 1.4.2 Ccdc66-Genexpression in Retina und Gehirn ..................................................... 21 1.5 Transkriptionsunterschiede in Retina und Gehirn der Ccdc66-/--Maus ..................... 23 1.5.1 Genexpression in Retina und Gehirn der Ccdc66-/--Maus .................................. 23 1.5.2 Kandidatengene für weitere Untersuchungen ..................................................... 25 2 Aufgabenstellung und Zielsetzung ................................................................. 26 3 Material und Methoden .................................................................................. 28 3.1 Versuchstiere ............................................................................................................ 28 3.2 Material ...................................................................................................................... 28 3.2.1 Enzyme ................................................................................................................ 28 3.2.2 Größenstandards ................................................................................................ 28 3.2.3 Oligonukleotide .................................................................................................... 29 3.2.4 Antikörper ............................................................................................................ 29 3.2.5 Reaktionskits ....................................................................................................... 30 3.2.6 Geräte .................................................................................................................. 30 3.2.7 Chemikalien und Lösungen ................................................................................. 30 3.2.8 Software .............................................................................................................. 32 3.2.9 Datenbanken ....................................................................................................... 32 3.3 Methoden .................................................................................................................. 32 3.3.1 Genotypisierung .................................................................................................. 32 1 3.3.1.1 DNA-Isolation .............................................................................................. 32 3.3.1.2 DNA-Amplifikation mittels PCR ................................................................... 33 3.3.1.3 Agarosegel-Elektrophorese ......................................................................... 34 3.3.2 Gewebepräparation ............................................................................................. 36 3.3.3 Anfertigung der Gefrierschnitte ........................................................................... 36 3.3.4 Herstellung der RNA-Sonden .............................................................................. 36 3.3.4.1 RNA-Isolation .............................................................................................. 37 3.3.4.2 Herstellung von cDNA mittels reverser Transkription ................................. 37 3.3.4.3 Amplifikation der Sondenvorlagen mittels PCR .......................................... 38 3.3.4.4 Elektrophoretische Auftrennung der Sondenvorlagen ................................ 40 3.3.4.5 Aufreinigung der Sondenvorlagen ............................................................... 41 3.3.4.6 Herstellung Digoxigenin-markierter RNA-Sonden ....................................... 42 3.3.5 In-situ-Hybridisierung .......................................................................................... 46 4 Ergebnisse .................................................................................................... 49 4.1 RNA-Expression in der Maus-Retina ........................................................................ 49 4.1.1 Serpina3n-RNA-Expression in der Maus-Retina................................................. 49 4.1.2 Gm12541-RNA-Expression in der Maus-Retina ................................................. 50 4.1.3 Antxr2-RNA-Expression in der Maus-Retina ....................................................... 52 4.1.4 Pcolce-RNA-Expression in der Maus-Retina ...................................................... 53 4.1.5 Gadd45b-RNA-Expression in der Maus-Retina .................................................. 54 4.1.6 Edn2-RNA-Expression in der Maus-Retina ......................................................... 55 4.1.7 Optc-RNA-Expression in Retina und Ziliarkörper der Maus ............................... 56 4.1.8 Rpe65-RNA-Expression in der Maus-Retina ...................................................... 64 4.1.9 Rho-RNA-Expression in der Maus-Retina .......................................................... 65 4.1.10 Zusammenfassung der Ergebnisse (Retina) ....................................................... 66 4.2 Zerebrale RNA-Expression der untersuchten Gene in der Maus ............................. 68 4.2.1 Optc-RNA-Expression im Gehirn der Maus ........................................................ 68 4.2.2 Pcolce-, Antxr2- und Gm12541-Expression im Gehirn der Maus ....................... 70 4.2.3 Serpina3n-, Edn2- und Gadd45b-Expression im Gehirn der Maus .................... 70 4.2.4 Zusammenfassung der Ergebnisse (Gehirn) ...................................................... 73 5 Diskussion ..................................................................................................... 75 5.1 Gene der zellulären Stressantwort ............................................................................ 76 2 5.2 Gadd45b im Kontext der Neuroprotektion ................................................................ 79 5.3 Gene der extrazellulären Matrix ................................................................................ 80 5.3.1 Auswirkung erhöhter retinaler Optc-Expression .................................................. 81 5.3.2 Antxr2- und Pcolce-Expression und Umbauprozesse der Extrazellularmatrix ... 85 5.4 Optc-, Edn2- und Serpina3n-Expression im Gehirn .................................................. 88 5.5 Schlussfolgerung und Ausblick ................................................................................. 89 6 Zusammenfassung ........................................................................................ 93 7 Literaturverzeichnis ....................................................................................... 96 3 Abkürzungsverzeichnis * Antxr2 anthrax toxin receptor 2 AP alkalische Phosphatase ATP Adenosintriphosphat BCIP 5-Brom-4-chlor-3-indoxylphosphat βgeo β-Galaktosidase/Neomycin-Fusionsgen BMP-1 bone morphogenetic protein 1 bp Basenpaare BSA bovines Serumalbumin Ca Calcium Ccdc66 coiled coil domain containing 66 CA Cornu Ammonis Ccdc66-/- coiled coil domain containing 66-defizienter Mausstamm Ccdc66+/+ coiled coil domain containing 66-Wildtyp cDNA complementary (komplementäre) DNA Cmg2 capillary morphogenesis gene-2 CUB complement C1r/C1s, UEGF, BMP-1 CTP Cytidintriphosphat DEPC Diethylpyrocarbamid DG Gyrus dendatus DIG Digoxigenin DNA Desoxyribonukleinsäure dNTP Desoxyribonukleosidtriphosphat dT Desoxythymin E embryonaler Tag Edn2 endothelin 2 EDTA Ethylendiamintretraessigsäure EPL external plexiform layer (externe plexiforme Schicht) FGF-2 fibroblast growth factor 2 Fgf2os fibroblast growth factor 2 opposite strand Fwd forward Gadd45b growth arrest and DNA-damage inducible, beta GAG Glykosaminoglykan GCL ganglion cell layer (Ganglienzellschicht) GDP Guanosindiphosphat GL glomerular
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