The role of LIN9 in mouse development [Elektronische Ressource] / vorgelegt von Nina Reichert

Nina Reichert

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

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


















E ing er e ic ht 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 D R M C o m p l e x i n 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 P l a sm i d l i st ............................................................................................................................. 27
2.1.7 Antibodies .............................................................................................................................. 28
2.1.8 Cell Lines & Cell Culture Media............................................................................................... 28
2.1.9 Bacterial S t r a i n s ..................................................................................................................... 29
2.1.10 Mouse Strains & ES Cell Lines ............................................................................................... 29
2.1.11 Microscopic equipment ........................................................................................................... 29
2.2 Methods ............................................................................................................................ 30
2.2.1 C e l l C u l t u r e ............................................................................................................................ 30
2.2.1.1. Generation of MEFs ................................................................................................................................... 30
2.2.1.2. P a s s a g e i n g o f C e l l s ................................................................................................................................... 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. D e t e r m i n a t i o n o f C e l l C y c l e P h a s e s .......................................................................................................... 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. T a i l P C R s ( b a s i c p r o t o c o l ) ......................................................................................................................... 36
2.2.2.10. Reverse Transcription ................................................................................................................................ 36
2.2.2.11. Quantitative PCR ...