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Charakterisierung Der Funktionellen Bedeutung Der Mikrorna-34A Während Der Entwicklung Und Der Tumorgenese Mittels Eines Mausmodells

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Charakterisierung Der Funktionellen Bedeutung Der Mikrorna-34A Während Der Entwicklung Und Der Tumorgenese Mittels Eines Mausmodells Charakterisierung der funktionellen Bedeutung der mikroRNA-34a während der Entwicklung und der Tumorgenese mittels eines Mausmodells Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie an der Universität Duisburg-Essen vorgelegt von Theresa Thor aus Gelsenkirchen Juni 2012 Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden im hämatologisch- onkologischen Labor der Kinderklinik III der Uniklinik Essen durchgeführt. 1. Gutachter: Prof. Dr. med. Johannes H. Schulte 2. Gutachter: PD. Dr. rer. nat. Wiebke Hansen 3. Gutachter: Prof. Dr. med. Olaf Witt Vorsitzender des Prüfungsausschusses: Prof. Dr. Michael Ehrmann Tag der mündlichen Prüfung: 16.11.2012 Für meine Eltern Inhaltsverzeichnis Inhaltsverzeichnis 1 Einleitung ........................................................................................................................... 1 1.1 Die Entdeckung der mikroRNAs ................................................................................. 1 1.2 Biogenese der mikroRNAs .......................................................................................... 2 1.3 Die Funktion von mikroRNAs ..................................................................................... 3 1.4 mikroRNAs während der Tumorgenese ...................................................................... 4 1.4.1 miRNAs als Onkogene ......................................................................................... 4 1.4.2 miRNAs als Tumorsuppressorgene ...................................................................... 7 1.5 Die miR-34- Familie .................................................................................................. 10 1.6 MiR-34a – Funktionen und Zielgene ......................................................................... 11 1.7 Die Bedeutung von miRNAs als Biomarker und in zukünftigen Therapien ............. 15 1.7.1 miRNA Mimics .................................................................................................. 15 1.7.2 Anti-mikroRNAs ................................................................................................ 16 1.8 Das Medulloblastom .................................................................................................. 18 1.8.1 Klassifizierung der Medulloblastome ................................................................ 19 1.8.2 Etablierte Mausmodelle des Medulloblastoms .................................................. 22 1.9 Zielsetzung der Arbeit ................................................................................................ 25 2 Material und Methoden .................................................................................................... 26 2.1 Materialien ................................................................................................................. 26 2.1.1 Chemikalien ....................................................................................................... 26 2.1.2 Geräte ................................................................................................................. 28 2.1.3 Verbrauchsmaterialien ....................................................................................... 29 2.1.4 Kits ..................................................................................................................... 29 2.1.5 Enzyme ............................................................................................................... 30 2.1.6 Antikörper .......................................................................................................... 30 2.1.7 Plasmide ............................................................................................................. 31 2.1.8 Oligonukleotide .................................................................................................. 32 2.1.9 Größen- und Molekulargewichtsmarker ............................................................ 33 2.1.10 Mauslinien .......................................................................................................... 33 2.1.11 Häufig verwendete Medien und Lösungen ........................................................ 34 2.1.11.1 Lösungen für molekularbiologische Arbeiten ............................................. 34 2.1.11.2 Lösungen für proteinbiochemische Arbeiten ............................................... 34 2.1.11.3 Medien für die Bakterienanzucht ................................................................. 36 I Inhaltsverzeichnis 2.1.11.4 Medien für die Zellkultur ............................................................................. 37 2.2 Methoden ................................................................................................................... 38 2.2.1 Molekularbiologische Methoden........................................................................ 38 2.2.1.1 Agarose-Gelelektrophorese von DNA- Fragmenten ................................... 38 2.2.1.2 Plasmid-DNA Aufreinigung im großen Maßstab (Maxi- Präparation) ....... 38 2.2.1.3 Bestimmung der RNA- und DNA-Konzentration ....................................... 38 2.2.1.4 Isolierung genomischer DNA aus Schwanzspitzenbiopsien der Maus ........ 39 2.2.1.5 Polymerasekettenreaktion (PCR) (modifiziert nach Saiki et al., 1985) ....... 39 2.2.1.6 Isolierung von Gesamt- RNA ...................................................................... 41 2.2.1.7 cDNA-Synthese ........................................................................................... 41 2.2.1.8 Quantitative Real-Time PCR ....................................................................... 42 2.2.2 Zellbiologische Methoden .................................................................................. 42 2.2.2.1 Anzucht und Passagieren von Zellen ........................................................... 42 2.2.2.1 Auftauen und Einfrieren von Zellen ............................................................ 42 2.2.2.2 Zellzahlbestimmung ..................................................................................... 43 2.2.2.3 Transiente Transfektion ............................................................................... 43 2.2.2.4 Bestimmung der Zellviabilität nach Transfektion ....................................... 44 2.2.2.5 Bestimmung der Zellproliferation nach Transfektion.................................. 44 2.2.2.6 Bestimmung der Apoptose nach Transfektion ............................................. 45 2.2.2.7 Seneszenz- Assay nach Transfektion ........................................................... 45 2.2.2.8 Zellzyklus-Analyse mittels Durchflusszytometrie (FACS-Analyse) ........... 45 2.2.3 Proteinbiochemische Methoden ......................................................................... 46 2.2.3.1 Herstellung von Proteinlysaten .................................................................... 46 2.2.3.2 Bestimmung von Proteinkonzentrationen .................................................... 46 2.2.3.3 Natriumdodecylsulfat-Polyacrylamid-Gelelektrophorese (SDS-PAGE)..... 47 2.2.3.4 Elektrophoretischer Protein-Transfer (Western Blot) .................................. 48 2.2.3.5 Immunologischer Nachweis von Proteinen (Immundetektion) ................... 49 2.2.4 Generierung transgener miR-34a knockout -Mäuse ............................................ 50 2.2.4.1 Isolierung genomischer miR-34a DNA und Klonierung des pMirn34a- Vektors ......................................................................................................... 50 2.2.4.2 Transfektion der ES-Zellen .......................................................................... 50 2.2.4.3 Generierung der chimären Mäuse ................................................................ 51 2.2.4.4 Aufbau der kongenen Mauslinie .................................................................. 51 2.2.5 Tierexperimentelles Arbeiten ............................................................................. 51 2.2.5.1 Zucht und Haltung ....................................................................................... 51 II Inhaltsverzeichnis 2.2.5.2 Entnahme von Schwanzbiopsien und Identifikation der Tiere .................... 52 2.2.5.3 Tötung von Versuchstieren .......................................................................... 52 3 Ergebnisse ........................................................................................................................ 53 3.1 Generierung der Mauslinie ......................................................................................... 53 3.1.1 Klonierung des pMirn34a-Targetingvektors zur homologen Rekombination in embryonale Stammzellen ................................................................................................. 53 3.1.2 Generierung chimärer MiR-34a-Mäuse ............................................................. 54 3.1.2.1 Elektroporation und Selektion der ES-Zellen .............................................. 55 3.1.2.2 Nachweis der homologen Rekombination ................................................... 55 3.1.2.3 Generierung chimärer Mäuse und Aufbau der MiR-34a-ko Mauslinie ....... 56 3.1.3 Genotypisierung ................................................................................................. 57 3.1.4 Expression der miR-34a in der Maus ................................................................. 57 3.2 Phänotypisierung der knockout-Maus in der ‚German Mouse Clinic‘ .....................
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