University of Bath PHD Characterisation of two mouse mutant lines generated by transgene insertional mutagenesis Paisley, Derek John Award date: 2000 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 CHARACTERISATION OF TWO MOUSE MUTANT LINES GENERATED BY TRANSGENE INSERTIONAL MUTAGENESIS Submitted by Derek John Paisley for the degree of PhD of the University of Bath 2000 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with the author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis and no information derived from it may be published without the prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. 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Box 1346 Ann Arbor, Ml 48106-1346 UNIVERSITY OF BATH LIBRARY - 3 or.T 2001 R-V. 1 Characterisation of Two Mouse Mutant Lines Generated By Transgene Insertional Mutagenesis Derek John Paisley, PhD University of Bath, 2000 ABSTRACT Some 74 lines of transgenic mice were screened for insertional mutant phenotypes using a two-step breeding strategy. A number of interesting developmental mutations were identifed and confirmed as resulting from transgene insertional mutagenesis. The further characterisation of two of these lines is described. The Ann line displayed a severe ataxic phenotype, leading to death at an early age. Histological analysis of the brains of these mice revealed abnormal neuronal patterning in the cerebellum, cerebral cortex and hippocampus. The site of transgene insertion was mapped on Chromsome 4 by a combination of Fluorescence In-Situ Hybridisation (FISH) and backcross linkage mapping. The expression of several candidate genes mapping close to the transgene was tested. Expression of the mouse disabled ( mdabl) gene was completely ablated in the brains of Ann mutants. Several mutant alleles of this gene have already been described that result in a phenotype indistinguishable from that seen in Ann mutants. The Ann mutant thus represents a new member of a pre-existing allelic series at this locus. 2 Male homozygotes of the Ob transgenic line have never been identified. That a significant proportion of Ob transgenic males did not breed strongly suggested that this line was affected by male sterility. Non-breeding males were confirmed as homozygous transgenics by FISH on interphase chromosome spreads. Additionally, histological studies showed abnormalities in testicular morphology as well as a reduction in a subset of spermatogenic cell types. Again the site of transgene insertion was mapped by FISH and linkage mapping. No strong candidate genes are present around the site of transgene integration on chromosome 8 suggesting that a novel mutation could be present in the Ob line. I wish to dedicate this thesis to my parents, Kathleen and Alexander Paisley. 4 ACKNOWLEDGMENTS Firstly, I would like to give my warmest thanks to my supervisor Dr. Andrew Ward. Not just for his excellent supervision but also for taking me on when I most needed a break. The contribution made by Dr. Bill Bennett to my PhD cannot be understated. Bill not only led the way but also set an extremely high standard that I could only hope to live up to. Thanks to everyone else in lab 0.76 and the rest of the Developmental Biology Programme for generating an enjoyable, challenging and often exhilarating atmosphere in which to work. I must also mention the staff of the University of Bath animal facility for their camaraderie. I thank my Mother and Father for their unstinting personal support. I also cannot forget Claire Adams for sharing the mostly good times with me during our tenure in Bath. A special mention must go to Eamon Mallon for putting up with me and still managing to be my favourite ever flatmate/lunchtime companion. More than thanks go to Lucy Allen for providing me with the best ever excuse to take a break from writing-up. Thanks to Benjamin Cravatt for providing the faah cDNA clone and to Chris Walsh and Jonathan Cooper for the mdabl clones. Janet Smith provided guidance for perfusion fixation. Thanks also to Professor Chris Graham who assisted me in re-deriving a number of lines in Bath. Muriel Lee provided technical advice for FISH and G-banding and also an expert opinion on chromosomal assignments. ABBREVIATIONS A adenosine bp base pairs BSA bovine serum albumin, fraction V C cytosine cDNA complimentary DNA CCD centrally conserved domain (Igf2/H19 intergenic region) CCD charge-coupled device (camera) Chr chromosome cM centimorgan(s) CNS central nervous system DEPC diethylpyrocarbonate DMR Igf2 5' differentially methylated region DNA deoxyribonucleic acid EDTA ethylene diamine tetra-acetic acid, sodium salt ENU ethylnitrosourea ES cells embryonic stem cells EtBr ethidium bromide EtOH ethanol FITC fluorescein isothiocyanate H&E haematoxylin and eosin IAP intracistemal A particle m gene encoding insulin-like growth factor-II IMS industrial methylated spirit kb kilobase(s) LB Luria-Bertani medium Luc luciferase coding region M molar MOPS 3-[N-Morpholino]propanesulfonic acid mRNA messanger ribonucleic acid nt mucleotide(s) NTD neural tube defect OD optical density PAC phage (PI) artificial chromosome PBS phosphate buffered saline PCR polymerase chain reaction QTL quantitative trait locus RFLP restriction fragment length polymorphism RI recombinant inbred rpm revolutions per minute Rnase ribonuclease RT reverse transcriptase SDS sodium dodecyl sulfate SSC 150mM NaCl, 15mM Na citrate SSCT 4xSSC containing 0.05% Tween-20 SSCTM SSCT + 5% non-fat milk SSPE 20mM NaHjPO^ 0.3M NaCl, 2mM EDTA SV40 simian virus 40 TBE tris/boric acid/EDTA TE lOmM Tris.HCl pH 8.0, ImM EDTA YAC yeast artificial chromosome 7 TABLE OF CONTENTS ABSTRACT......................................................................................................................................... 1 ACKNOWLEDGMENTS................................................................................................................... 4 ABBREVIATIONS.............................................................................................................................. 5 TABLE OF CONTENTS....................................................................................................................7 LIST OF TABLES............................................................................................................................ 15 LIST OF FIGURES.......................................................................................................................... 16 CHAPTER 1: INTRODUCTION ........................................................................................................................... 18 1.1 T he L a b o r a t o r y M o u se A s A M o del S y s t e m ................................................................................... 19 1.2 T ools Fo r st u d y in g Th e M o u se Gen o m e - Gen o ty pe b a s e d A n a l y s is ...............................20 1.2.1 Gene targeting ........................................................................................................................20 1.2.2 CreAox P technology .............................................................................................................23 1.3 T ools F o r St u d y in g T h e M o u se Gen o m e - P h eno ty pe B a s e d A n a l y sis ............................ 25 1.3.1 Gene traps .............................................................................................................................. 25 1.3.2 Transgene insertional mutagenesis ..................................................................................... 26 1.3.3 Mutagenic agents ...................................................................................................................2