THE ROLE OF LISSENCEPHALY-1 PROTEIN IN MALE GERM CELL DIFFERENTIATION Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Nadja Drusenheimer aus Remscheid Göttingen 2009 D 7 Referent: Prof. Dr. med Dr. h.c. W. Engel Korreferentin: Prof. Dr. S. Hoyer-Fender Tag der mündlichen Prüfung: Table of Contents I TABLE OF CONTENTS TABLE OF CONTENTS ----------------------------------------------------------------------- I ABBREVIATIONS ----------------------------------------------------------------------------- V 1. INTRODUCTION ----------------------------------------------------------------------------1 1.1 Pafah1b1/Lis1 is an evolutionary conserved gene---------------------------------------------------------- 1 1.2 Mutations in LIS1 gene cause classical lissencephaly ----------------------------------------------------- 2 1.3 Expression and function of Lis1 ------------------------------------------------------------------------------- 3 1.4 Generation of the gene trap line L39GT/GT ------------------------------------------------------------------- 6 1.5 Analysis of the gene trap line L39GT/GT----------------------------------------------------------------------- 7 1.6 Objectives of this study------------------------------------------------------------------------------------------ 9 2. MATERIALS AND METHODS --------------------------------------------------------- 10 2.1 Materials ----------------------------------------------------------------------------------------------------------10 2.1.1 Chemicals ---------------------------------------------------------------------------------------------------10 2.1.2 Solutions, buffers and media ----------------------------------------------------------------------------13 2.1.3 Laboratory materials -------------------------------------------------------------------------------------16 2.1.4 Sterilisation of solutions and equipments-------------------------------------------------------------17 2.1.5 Media, antibiotics and agar-plates ---------------------------------------------------------------------17 2.1.5.1 Media for bacteria-----------------------------------------------------------------------------------17 2.1.5.2 Media for cell culture -------------------------------------------------------------------------------17 2.1.5.3 Antibiotics---------------------------------------------------------------------------------------------19 2.1.5.4 IPTG/X-Gal plates-----------------------------------------------------------------------------------19 2.1.6 Bacterial strain---------------------------------------------------------------------------------------------19 2.1.7 Plasmids -----------------------------------------------------------------------------------------------------19 2.1.8 Synthetic oligonucleotides -------------------------------------------------------------------------------20 2.1.9 cDNA probes for Northern blotting--------------------------------------------------------------------24 2.1.10 Eukaryotic cell lines -------------------------------------------------------------------------------------24 2.1.11 Mouse strains ---------------------------------------------------------------------------------------------25 2.1.12 Antibodies -------------------------------------------------------------------------------------------------25 2.1.13 Enzymes----------------------------------------------------------------------------------------------------25 2.1.14 Kits----------------------------------------------------------------------------------------------------------26 2.1.15 Instruments -----------------------------------------------------------------------------------------------26 2.2 Methods -----------------------------------------------------------------------------------------------------------27 2.2.1 Isolation of nucleic acids ---------------------------------------------------------------------------------27 2.2.1.1 Isolation of plasmid DNA --------------------------------------------------------------------------27 2.2.1.2 Isolation of genomic DNA from murine tail biopsies -----------------------------------------29 2.2.1.3 Isolation of total RNA from tissue samples and cultured cells------------------------------29 2.2.2 Determination of nucleic acid concentration---------------------------------------------------------30 2.2.3 Gel electrophoresis ----------------------------------------------------------------------------------------30 2.2.3.1 Agarose gel electrophoresis of DNA--------------------------------------------------------------31 2.2.3.2 Agarose gel electrophoresis of RNA--------------------------------------------------------------31 2.2.3.3 SDS-PAGE for separation of proteins-----------------------------------------------------------31 2.2.4 Isolation of DNA fragments after gel electrophoresis ----------------------------------------------32 Table of Contents II 2.2.5 Enzymatic modifications of DNA-----------------------------------------------------------------------32 2.2.5.1 Digestion of DNA using restriction enzymes ---------------------------------------------------32 2.2.5.2 Ligation of DNA fragments------------------------------------------------------------------------33 2.2.5.3 Phenol-chloroform extraction and ethanol precipitation ------------------------------------33 2.2.5.4 TA-Cloning -------------------------------------------------------------------------------------------33 2.2.5.5 Filling-up reaction -----------------------------------------------------------------------------------34 2.2.6 Transformation of competent E.coli bacteria--------------------------------------------------------34 2.2.7 Polymerase Chain Reaction (PCR) --------------------------------------------------------------------35 2.2.7.1 PCR amplifications of DNA fragments----------------------------------------------------------35 2.2.7.2 Reverse transcription PCR (RT-PCR) ----------------------------------------------------------36 2.2.7.3 Quantitative Real-Time PCR----------------------------------------------------------------------36 2.2.7.4 Quantitative Real-Time RT-PCR-----------------------------------------------------------------38 2.2.7.5 PCR-based “Genome-Walking” ------------------------------------------------------------------39 2.2.8. Protein and biochemical methods ---------------------------------------------------------------------40 2.2.8.1 Isolation of total proteins---------------------------------------------------------------------------40 2.2.8.2 Determination of protein concentration---------------------------------------------------------40 2.2.9 Blotting techniques ----------------------------------------------------------------------------------------41 2.2.9.1 Southern blotting of DNA onto nitrocellulose filter-------------------------------------------41 2.2.9.2 Northern blotting of RNA onto nitrocellulose filter-------------------------------------------41 2.2.9.3 Western blotting of protein onto PVDF membrane-------------------------------------------42 2.2.10 "Random Prime" method for generation of 32P-labeled DNA ---------------------------------43 2.2.11 Hybridisation of nucleic acids -------------------------------------------------------------------------43 2.2.12 Non-radioactive dye terminator cycle sequencing-------------------------------------------------43 2.2.13 Histological techniques----------------------------------------------------------------------------------44 2.2.13.1 Tissue preparation for paraffin-embedding --------------------------------------------------44 2.2.13.2 Sections of the paraffin block --------------------------------------------------------------------45 2.2.13.3 Tissue preparation for cryopreservation ------------------------------------------------------45 2.2.13.4 Cryosectioning --------------------------------------------------------------------------------------45 2.2.13.5 Hematoxylin & Eosin staining of histological sections--------------------------------------45 2.2.13.6 LacZ staining of tissue sections------------------------------------------------------------------46 2.2.13.7 Tissue preparation for electron microscopy --------------------------------------------------46 2.2.14 Indirect immunohistochemistry-----------------------------------------------------------------------46 2.2.15 Generation of transgenic mice-------------------------------------------------------------------------47 2.2.15.1 Preparation of DNA for pronuclear microinjection-----------------------------------------47 2.2.16 Determination of sperm parameters -----------------------------------------------------------------47 2.2.16.1 Sperm count in epididymis, uterus and oviduct----------------------------------------------47 2.2.16.2 Sperm motility --------------------------------------------------------------------------------------48 2.2.17 Eukaryotic cell culture methods ----------------------------------------------------------------------48 2.2.17.1 Cell culture conditions ----------------------------------------------------------------------------48 2.2.17.2 Preparation of MEFs feeder layers -------------------------------------------------------------48 2.2.17.3 Trypsinization of eukaryotic cells---------------------------------------------------------------49 2.2.17.4 Cryopreservation and thawing of eukaryotic cells ------------------------------------------49 2.2.17.5 Transfection of eukaryotic cells with