University of Bath PHD Characterisation of two developmentally important genes mutated by transgene insertion in the laboratory mouse Bennett, William R. Award date: 1999 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 1 CHARACTERISATION OF TWO DEVELOPMENTALLY IMPORTANT GENES MUTATED BY TRANSGENE INSERTION IN THE LABORATORY MOUSE. Submitted by William R. Bennett for the degree of PhD of the University of Bath 1999 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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Bennett, PhD University of Bath, 1999 ABSTRACT A large number of transgenic mouse lines have been screened for post-natal mutant phenotypes by breeding to homozygosity, along with partial screening for pre- and peri-natal phenotypes during Caesarean re­ derivation of the lines in Bath. Various developmental abnormalities were observed, some of which have been caused by disruption of somatic gene function by transgene insertion. Two lines were selected for further characterisation. In the Harry transgenic line, the transgene has integrated close to the SoxlO locus on chromosome 15. SoxlO encodes a transcription factor which is expressed in a subset of neural crest cells during development and continues to be expressed in peripheral glia and oligodendrocytes during adult life. Harry homozygous mutants display a recessive phenotype which features limited skin pigmentation and toxic megacolon caused by a lack of enteric neurons. This is caused by transgene-induced disruption of correct function of the SoxlO gene product in neural crest cells. SoxlODom (dominant megacolon) is a spontaneous mouse mutant which encodes a truncated form of the SoxlO protein. SoxlODom/SoxlODom homozygotes are embryonic lethal by E13, but heterozygotes display essentially the same phenotype observed in Harry mice which are homozygous for the transgene. Harry mutants express wild-type levels of full-length SoxlO mRNA in the CNS, however, and there is no disruption either within the SoxlO gene itself or within 2kb of the start or end of the transcribed region. 3 In the Holly transgenic line, homozygosity for the transgene causes acute angle-closure glaucoma to develop shortly after the eyes open around day 12 post-partum. Insertion of the transgene into proximal chromosome 1 appears to have disrupted the expression of Col9al in a tissue-specific manner. Col9al encodes the a l chain of type IX collagen, a heterotrimeric protein thought to be important in mediating the interaction between structural collagen fibrils and the extracellular matrix in which they are embedded. 4 DEDICATION This thesis is dedicated to Dr Deb Speden, for faithfully holding my ropes, both literally and metaphorically, and to "Pinky and the Brain", for inspiration. "Both by virtue of its massiveness, and by the ubiquity and variety of tissues to which it gives rise, [the neural crest] would merit the status of a fourth germ layer were it not that it is above all from the study of its neural crest component that the limitations of the germ-layer concept in vertebrate embryology has been most strikingly demonstrated." (Newth, 1951) 18th century Japanese piebald mice (Chingan-sodategusa, author unknown) "Dear Mr Bennett, I am responding to your letter dated 15th March 1999, requesting permission to use Warner Bros, materials, specifically a Pinky and the Brain GIF image from a Warner Bros website (the "Materials") in your PhD thesis. While we greatly appreciate your interest in using the Materials, Warner Bros, has a long standing policy against specifically granting anyone the rights that you seek. This is why I must deny your request for use. We wish you the very best of luck with your academic career. Kind Regards, Rick Kent, Manager, Legal Affairs, Warner Bros. Online" 5 ACKNOWLEDGMENTS I would like to thank Dr William J. Pavan (NHGRI, NIH, Bethesda, Maryland, USA) for helpful discussion and suggestions on the use of linkage mapping in transgenic insertional mutants. The mouse SoxlO cDNA plasmids were a kind gift of Dr Michelle Southard-Smith (Pavan lab). I would also like to thank Dr Gerd Scherer (Institute of Human Genetics and Anthropology, Freiburg, Germany) for generously sharing unpublished data (the mouse SoxlO gene structure and restriction map). Prof Michel Goosens and Dr Veronique Pingault (Hopital Henri Mondor, Creteil, France) originally derived and provided me with the SoxlO RT-PCR protocol and primer sequences. The Dvll genomic clone was a gift of Dr Daniel Sussman (University of Maryland School of Medicine, Baltimore, USA), and the Lifr genomic clone was a gift of Dr Ian Chambers (MRC Human Genetics Unit, Western General Hospital, Edinburgh). The CoWal probes pMCol9al-l and pMCol9al-2 were provided by Prof Eero Vuorio (University of Turku, Turku, Finland). Transfer of the Harry line transgene onto different genetic backgrounds as congenic strains and subsequent breeding to homozygosity was carried out by Prof Chris Graham (Department of Zoology, University of Oxford), under whose aegis all transgenic lines in this study were originally derived and to whom I am deeply indebted. I am eternally grateful to Dr Muriel Lee (MRC Human Genetics Unit, Western General Hospital, Edinburgh) for teaching me how to perform FISH, and for giving me her expert "best guess" for chromosomal identities in the G-banding experiments. I would like to acknowledge the expert assistance and hard work of animal house technicians both in Bath and Oxford. Finally, I would like to thank my supervisor, Dr. Andrew Ward, for giving me the freedom to pursue those avenues I thought most interesting, my fellow transgenic mutant investigator Derek Paisley for discussion and for mousing above and beyond the call and everyone else in lab 0.76 for providing a stimulating and fun environment to work in. This work was funded by the School of Biology & Biochemistry at University of Bath. 7 TABLE OF CONTENTS LIST OF TABLES.......................................................................................................11 LIST OF FIGURES.................................................................................................... 12 ABBREVIATIONS...................................................................................................14 CHAPTER ONE:........................................................................................................17 INTRODUCTION........................................................................................................17 Background ................................................................................................................................................ 17 Mouse mutants ...........................................................................................................................................21 Alternative approaches to mutagenesis in the mouse ........................................................................30 Summary ..................................................................................................................................................... 33 The future?.................................................................................................................................................33 Aims of the project ...................................................................................................................................