The Role of LIN9 in Mouse Development Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Nina Reichert aus Marburg an der Lahn Würzburg, 2008 Eingereicht am: ............................................................................................ Mitglieder der Promotionskommission: Vorsitzender: 1. Gutachter: Prof. Dr. Stefan Gaubatz 2. Gutachter: Prof. Dr. Thomas Brand Tag des Promotionskolloquiums: ............................................................................................ Doktorurkunde ausgehändigt am: ............................................................................................ Für meine Familie & Oliver Content I 1 INTRODUCTION ........................................................................................................................1 1.1 Cell Cycle & its Regulators ...................................................................................................1 1.1.1 Mammalian Cell Cycle .............................................................................................................. 1 1.1.2 Cyclins & CDKs ........................................................................................................................ 2 1.1.3 pRB/E2F Pathway .................................................................................................................... 4 1.2 pRB/E2F Complexes in Model Organisms ...........................................................................5 1.2.1 DRM Complex in C. elegans ..................................................................................................... 5 1.2.2 Myb-MuvB(MMB) & dREAM Complexes in Drosophila .............................................................. 6 1.2.3 Human LIN Complex & its Core Component LIN-9 .................................................................... 7 1.3 Mouse Models for known Cell Cycle Regulators ................................................................. 10 1.3.1 Embryonic Development ......................................................................................................... 10 1.3.2 Essential LINC Members ........................................................................................................ 12 1.3.2.1. pRB/p107/130 ............................................................................................................................................ 12 1.3.2.2. E2F4 ........................................................................................................................................................... 15 1.3.2.3. B-MYB ........................................................................................................................................................ 16 1.4 Aim of this Project .............................................................................................................. 17 2 MATERIALS & METHODS....................................................................................................... 18 2.1 Materials ............................................................................................................................ 18 2.1.1 Chemical Stocks & Reagents.................................................................................................. 18 2.1.2 PCR & DNA/RNA Modifying Enzymes..................................................................................... 19 2.1.3 Molecular Kits & Protein/DNA/RNA Markers ............................................................................ 20 2.1.4 Buffers ................................................................................................................................... 20 2.1.4.1. General Buffers .......................................................................................................................................... 20 2.1.4.2. Buffers for Southern Blot ........................................................................................................................... 22 2.1.4.3. Buffers for Immunohistochemistry ............................................................................................................. 23 2.1.4.4. Buffers for Western Blot ............................................................................................................................. 24 2.1.5 Oligolist .................................................................................................................................. 25 2.1.6 Plasmidlist ............................................................................................................................. 27 2.1.7 Antibodies .............................................................................................................................. 28 2.1.8 Cell Lines & Cell Culture Media............................................................................................... 28 2.1.9 Bacterial Str ai ns ..................................................................................................................... 29 2.1.10 Mouse Strains & ES Cell Lines ............................................................................................... 29 2.1.11 Microscopic equipment ........................................................................................................... 29 2.2 Methods ............................................................................................................................ 30 2.2.1 Cell Culture ............................................................................................................................ 30 2.2.1.1. Generation of MEFs ................................................................................................................................... 30 2.2.1.2. Passageing of Cells ................................................................................................................................... 30 2.2.1.3. Freezing & Thawing of Cells ...................................................................................................................... 30 2.2.1.4. Transient Transfection of Plat-E Cells ....................................................................................................... 31 2.2.1.5. Infection of MEFs ....................................................................................................................................... 31 2.2.1.6. Tamoxifen Induction of Cre........................................................................................................................ 31 2.2.1.7. Growth Curve ............................................................................................................................................. 32 2.2.1.8. Determination of Cell Cycle Phases .......................................................................................................... 32 2.2.1.9. Culturing of Blastocysts ............................................................................................................................. 32 2.2.2 Molecular Methods ................................................................................................................. 32 Content II 2.2.2.1. Isolation of RNA from Cultured Cells......................................................................................................... 32 2.2.2.2. Isolation of RNA from Mouse Organs ....................................................................................................... 33 2.2.2.3. Isolation of Plasmid DNA from Bacteria .................................................................................................... 33 2.2.2.4. Isolation of DNA Fragments from Agarose Gels ....................................................................................... 33 2.2.2.5. Extraction of gDNA from Cells ................................................................................................................... 34 2.2.2.6. E x t r a c tion of gDNA in Small Scale ............................................................................................................ 34 2.2.2.7. Extraction of gDNA from Tails for Southern Blot ...................................................................................... 34 2.2.2.8. Standard Cloning Method .......................................................................................................................... 35 2.2.2.9. Tail PCRs (basic protocol) ......................................................................................................................... 36 2.2.2.10. Reverse Transcription ................................................................................................................................ 36 2.2.2.11. Quantitative PCR ....................................................................................................................................... 37 2.2.2.12. T a q M a n ...................................................................................................................................................... 37 2.2.2.13. Southern Blot with Radioactively Labeled Probes .................................................................................... 38 2.2.3 Immunohistochemical Methods ............................................................................................... 40 2.2.3.1. Preparation of Paraffin Sections ...............................................................................................................