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Strategies to Study Sex-Determining Genes in the Mouse Strategies to study sex-determining genes in the mouse Amy E. Johnson A thesis submitted to the University of London in fulfilment of the requirements for the degree of Doctor of Philosophy. May 2002 Division of Developmental Genetics, National Institute For Medical Research, The Ridgeway, Mill Hill, London, NW7 lAY. ProQuest Number: U643301 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643301 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. 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Box 1346 Ann Arbor, Ml 48106-1346 This thesis is dedicated to my immediate family, Janice, Clive, Molly, Matthew, Ben, Emma and my fiancé Geoff Table Of Contents ACKNOWLEDGEMENTS ............................................ / ABSTRACT ....................................................................................................................IV 1 CHAPTER 1- GENERAL INTRODUCTION....................................................... 1 lA SEX DETERMINATION......................................................................................1 1.1.1 Drosophila and C-ele^ans .................................................................................1 1.1.2 Birds................................................................................................................... 3 1.1.3 Mammals............................................................................................................4 1.1.4 Reptiles ..............................................................................................................4 E2 DIFFERENTIATION OF THE GONAD AND INTERNAL GENITALIA. ....................................................................................................................................5 1.2.1 Testis descent.....................................................................................................6 13 CELL LINEAGES...................................................................................................7 L4 SOMATIC CELL DIFFERENTIATION.......................................................... 10 13 INDENTIFICATION OF THE TESTIS DETERMINING GENE...............15 1.5.1 Biochemical Properties of SRY ..................................................................... 15 13 GENES REOUIRED FOR GONAD FORMATION.......................................18 1.6.1 WT-1................................................................................................................ 18 1.6.2 STERIODOGENIC FACTOR 1 (SFl)..........................................................20 1.6.3 LHX9 ...............................................................................................................21 1.6.4 M33.................................................................................................................. 22 L7 GENETIC CONTROL OF TESTIS FORMATION.......................................23 1.7.1 CAMPOMELIC DYSPLASIA AND S0X9 ................................................23 1.7.2 DM RTl............................................................................................................34 1.7.3 DESERT HEDGEHOG (Dhh).......................................................................35 1.7.4 TESTATIN..................................................................................................... 36 1.7.5 GATA-4 (see also AMH) ................................................................................37 1.7.6 FIBROBLAST GROWTH FACTOR 9 (FGF-9).........................................38 1.7.7 ANTI-MÜLLERIAN HORMONE (AMH)..................................................39 L8 SUMMARY ........................................................... 43 1^ GENES EXPRESSED DURING OVARIAN DEVELOPMENT................. 44 1.9.1 DOSAGE SENSITIVE SEX REVERSAL (DSS) and DAX-1 .................. 44 1.9.2 WNT-4 .............................................................................................................46 1.9.3 GDF-9 (‘Ooctyte-secreted growth factor’) ...................................................47 1.10 AIMS OF THIS TH ESIS.....................................................................................49 2 CHAPTER 2 - ENGINEERING A S0X9 CONDITIONAL MUTATION.. 51 2.1 INTRODUCTION................................................................................................. 51 2.1.1 Designing a conditional targeted mutation ................................................... 51 2.1.2 Inactivating Sox9 within the developing genital ridge ................................ 53 22 MATERIALS AND METHODS........................................................................56 2.2.1 Cloning tecliniques and reagents used to generate the Sox9 targeting construct .........................................................................................................................56 2.2.2 Bacteria used for amplification of Sox9 phage clone .................................. 56 2.2.3 Liquid culture of bacteriophage .....................................................................56 2.2.4 Isolation of bacteriophage X DNA.................................................................57 2.2.5 Preparation of Epicurian Coli XL 10-Gold Ultracompetent Cells. ..............57 2.2.6 Small and Large scale plasmid preparation. ................................................. 58 2.2.7 Restriction enzyme digestion of DNA ...........................................................58 2.2.8 Blue-white cloning Assay .............................................................................. 58 2.2.9 Quantification of nucleic acid concentrations ...............................................58 2.2.10 Electrophoretic separation of DNA ...............................................................59 2.2.11 Extraction of DNA ..........................................................................................59 2.2.12 Blunt-ending, phosphatasing and ligation of DNA fragments .................... 60 2.2.13 Partial endonuclease digestion of DNA .........................................................61 2.2.14 Colony Hybridization - Screening for recombinant plasmids .....................61 2.2.15 Generating double-stranded loxP and Fit oligonucleotides for use in the Sox9 conditional targeting mutation .............................................................................62 2.2.16 Double Stranded DNA Sequencing ...............................................................64 2.2.17 TISSUE CULTURE TECHNIQUES AND TRANSFECTION ASSAYS .........................................................................................................................65 23 RESULTS and DISCUSSION............................................................................67 2.3.1 Building the Sox9 conditional targeting vector ............................................67 2.3.2 Choosing a Reporter .......................................................................................71 2.3.3 PGK-Neo ......................................................................................................... 71 2.3.4 Testing the function of Sox9'^^^ in vitro before gene targeting .................. 73 2.3.5 Checking the expression of the MmGFP reporter in vitro prior to gene targeting .........................................................................................................................76 3 CHAPTER 3 - TARGETING THE S0X9 LOCUS INES CELLS USING THE CONSTRUCTPSOX9^^^^ ..................................................................................78 3J_ INTRODUCTION................................................................................................78 3.1.1 ES Cells........................................................................................................... 78 3.1.2 Gene targeting .................................................................................................78 3.1.3 Choosing the correct Embryonic Stem cell line ...........................................80 3.1.4 Differentiating Embryonic Stem cells in culture ..........................................80 32 MATERIALS AND METHODS........................................................................82 3.2.1 Cell Lines........................................................................................................ 82 3.2.2 Electroporation ................................................................................................83 3.2.3 Selection of transfected cells ......................................................................... 84 3.2.4 Extraction of DN A from ES cells (Death wish!) ........................................ 84 3.2.5 Southern blotting .............................................................................................85 3.2.6 Hybridisation ...................................................................................................86 3.2.7 Cre-mediated recombination between two loxP sites .................................. 86 3.2.8 Blastocyst injection of Sox9'"^'' targeted ES cells .................................. 87 3.2.9 Pseudopregnant recipients ............................................................................
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