The role of the PeBoW-complex in ribosome biogenesis and proliferation of mouse embryonic stem cells [Elektronische Ressource] / Iris Pfisterer

Iris Pfisterer

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 27 Mo

Extrait

The role of the PeBoW-complex
in ribosome biogenesis and proliferation
of mouse embryonic stem cells





Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München




vorgelegt von
Iris Pfisterer



5. November 2007



Diese Arbeit wurde am Institut für Klinische Molekularbiologie und Tumorgenetik des
GSF-Forschungszentrums für Umwelt und Gesundheit in München angefertigt.





























Erstgutachter: Prof. Dr. Dirk Eick
Zweitgutachter: Prof. Dr. Michael Schleicher

Tag der mündlichen Prüfung: 17.12.2007
Ehrenwörtliche Versicherung

Ich versichere hiermit ehrenwörtlich, dass die Dissertation von mir selbstständig und
ohne unerlaubte Beihilfe angefertigt worden ist.



Erklärung

Hiermit erkläre ich, dass die Dissertation nicht ganz oder in wesentlichen Teilen einer
anderen Prüfungskomission vorgelegt worden ist.
Ich erkläre weiterhin, dass ich mich nicht anderweitig einer Doktorprüfung ohne Erfolg
unterzogen habe.


München, im November 2007


Iris Pfisterer
















Table of content
TABLE OF CONTENT


SYNOPSIS .......................................................................................................................... 1
1 INTRODUCTION............................................................................................................ 3
1.1 Mouse embryonic stem cells ................................................................................ 3
1.1.1 Self-renewal of mouse embryonic stem cells .................................................... 4
1.1.2 Differentiation of mouse embryonic stem cells .................................................. 7
1.2 Proliferation and cell cycle ................................................................................... 8
1.2.1 Cell cycle and G1/S-phase checkpoint of mature cells ...................................... 9
1.2.2 Cell cycle of mouse embryonic stem cells ....................................................... 11
1.3 Nucleolus and cell cycle control ........................................................................ 13
1.3.1 Ribosome biogenesis in mammalian cells....................................................... 13
1.3.2 Link of nucleolar disruptions and cell cycle progression .................................. 14
1.4 The PeBoW-complex........................................................................................... 16
1.4.1 Pes1 ............................................................................................................... 18
1.4.2 Bop1 ............................................................................................................... 18
1.4.3 WDR12 ........................................................................................................... 19
1.5 Aim of the project ................................................................................................ 20
2 MATERIAL .................................................................................................................. 21
2.1 Cell lines............................................................................................................... 21
2.2 Media and supplements for cell culture............................................................. 21
2.3 siRNA-oligonucleotides ...................................................................................... 22
2.4 Antibodies............................................................................................................ 22
2.4.1 Primary antibodies .......................................................................................... 22
2.4.2 Secondary antibodies ..................................................................................... 23
2.5 Primer................................................................................................................... 23
2.6 Markers (protein and DNA) ................................................................................. 24
2.7 Kits ....................................................................................................................... 24
2.8 Disposables ......................................................................................................... 24
2.9 Chemicals, Reagents and Enzymes ................................................................... 25
3 METHODS ................................................................................................................... 27
3.1 Cell Culture .......................................................................................................... 27
3.1.1 Culture conditions of CGR8 cells .................................................................... 27
I Table of content
3.1.2 Culture conditions of NIH3T3 cells .................................................................. 27
3.1.3 Differentiation of CGR8 cells ........................................................................... 28
3.1.4 Freezing of CGR8 cells in liquid nitrogen ........................................................ 28
3.1.5 Transient transfection of CGR8 cells............................................................... 28
3.1.6 Transfection of CGR8 cells with siRNA ........................................................... 29
3.1.7 Transfection of NIH3T3 cells with siRNA......................................................... 29
3.1.8 Annealing of siRNA......................................................................................... 30
3.1.9 Counting of cells ............................................................................................. 30
3.2 Protein Analyses ................................................................................................. 30
3.2.1 Generation of antibodies ................................................................................. 30
3.2.1.1 Generation of polyclonal antibodies............................................................................ 30
3.2.1.2 Generation of monoclonal antibodies ......................................................................... 31
3.2.2 Western Blot Analysis ..................................................................................... 32
3.2.2.1 Preparation of protein extracts under denaturing conditions for SDS-PAGE .......... 32
3.2.2.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotting of proteins ..... 32
3.2.2.3 Hybridization with antibodies....................................................................................... 33
3.2.2.4 Stripping of membranes............................................................................................... 33
3.2.3 Native Gels ..................................................................................................... 34
3.2.3.1 Preparation of protein extracts under native conditions ............................................ 34
3.2.3.2 Native gel electrophoresis ........................................................................................... 34
3.2.4 Immunofluorescence....................................................................................... 35
3.3 RNA Analyses...................................................................................................... 35
3.3.1 Isolation of total RNA ...................................................................................... 35
3.3.2 Reverse transcription (RT) of RNA into cDNA................................................. 36
3.3.3 Quantitative real-time PCR analysis................................................................ 36
323.3.4 In vivo labeling of RNA with P-orthophosphate ............................................. 37
3.4 Cell Proliferation and Apoptosis ........................................................................ 38
3.4.1 Proliferation assay - cell count ........................................................................ 38
3.4.2 Proliferation assay - GIEMSA staining ............................................................ 38
3.4.3 Cell cycle analysis with propidium iodide (PI) staining..................................... 39
3.4.4 Cell cycle analysis with bromodeoxyuridine (BrdU) labeling............................ 39
3.4.5 Annexin V staining .......................................................................................... 40
4 RESULTS .................................................................................................................... 41
4.1 Expression, localization and complex formation of Pes1, Bop1 and
WDR12 in mouse embryonic stem cells ............................................................ 41
4.1.1 Generation of antibodies for the detection of murine Pes1, Bop1 and WDR12 41
4.1.2 Nucleolar localization of Pes1, Bop1 and WDR12 in CGR8 and NIH3T3 cells 43
II Table of content