DOCSLIB.ORG

The Isolation and Characterisation of the Novel Gene C53

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Isolation and Characterisation of the Novel Gene C53 Acknowledgements To begin, I would like to thank my supervisors. First of all, Rosa Beddington, her insight contributed greatly to this work and her passionate nature was an invaluable source of inspiration. Second, Steve Harrison, who provided thoughtful and thorough feedback that significantly improved this thesis. More than this, he made himself available to become my principle supervisor and was on hand throughout the main body of this work to answer my numerous questions. Finally, Sohaila Rastan and Vassilis Pachnis for their support and useful discussions. There are also many people I wish to thank for their invaluable contributions to the work. Special thanks to Evelyn Grau for carrying out the blastocyst injections. Graham Duddy for help and advice in tissue culture and our numerous technical discussions in and out of the lab. Davina Gale for invaluable support during the RDA, and Dave Michaelovich for help and advice on bioinformatics. Also, Juan Pedro Martinez Barbera and Alastair Morrison for reading my thesis and giving incisive feedback. Finally, everyone in the division of Mammalian Development and Comparative Genomics for their advice and support. I'd like to thank some wonderful friends for providing an important source of distraction. Whether it was driving across the country with me to jump in the sea or just getting me out of the lab to the relative sanity of the nearest pub, thanks for being there! Finally, to my family - Mom, Dad, and Sarah, for 26 years of love and support: how can I begin to thank you? The isolation and characterisation of the novel gene C53 Ross Kettleborough National Institute for Medical Research PhD. ProQuest Number: U641885 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 U641885 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. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The study of axis formation is crucial if we are to understand the formation of a functional body plan. In mouse, the first morphological marker of A-P pattern, the primitive streak, forms at the posterior of the embryo and drives a complex process called gastrulation, during which new germ layers are formed and a general body plan is generated that serves as a scaffold for the subsequent morphogenesis of the embryo. In order to isolate tissue specific genes that are involved in these symmetry breaking events a cDNA library derived from the endoderm of the 7.5dpc mouse embryo was produced. A screen was then performed that used sequence and expression information to further analyse the endoderm specific library. cDNA clones representing genes that are expressed in tissues intimately involved in the early patterning of the embryo (e.g. the visceral endoderm, the definitive endoderm and the node) were identified. This study focuses on C53, a novel gene that is expressed in the visceral endoderm, the node, and hematopoietic lineages of the early mouse embryo. A targeted mutation has been generated that results in homozygous null animals and causes embryonic lethality after 14.5dpc from severe anemia. In vitro differentiation of C53 null ES cells indicates that a de-regulation of hematopoietic transcription factors occurs when C53 is not present, and further analysis has shown that hematopoietic specific molecules are inappropriately expressed in the embryonic yolk sac and embryonic liver. This investigation indicates that the novel gene C53 is essential for the correct differentiation of certain hematopoietic lineages. Contents CHAPTER 1: GENERAL INTRODUCTION................................................. 10 1.1 Pre and post implantation mouse development and the embryonic axes..... 10 1.1.1 Early signs of asymmetry ..............................................................................10 1.1.2 A-P axis formation........................................................................................13 1.1.3 The VE is also implicated in A-P pattern formation .....................................15 1.2 Mouse haematopoiesis ............................................................................................ 18 1.2.1 Primitive haematopoiesis begins in the yolk sac ...........................................18 1.2.2 Definitive haematopoiesis is mediated by intra-embryonic sites .................. 19 1.3 Isolation and characterisation of novel genes involved in early patterning events...............................................................................................................................21 1.3.1 Strategies for the isolation of genes involved in early patterning .................21 1.3.2 ES cell technology in the study of gene function ..........................................24 1.4 The study of the novel gene C 53 .......................................................................... 26 CHAPTER 2: MATERIALS AND METHODS............................................... 29 2.1 Screening of the endoderm cDNA library ...........................................................29 2.1.1 Comparison of sequencing data from the endoderm library with sequence databases ................................................................................................................ 29 2.1.2 Dissection of peri-implantation mouse embryos ...........................................29 2.1.3 Whole mount in situ hybridisation using Digoxigenin-UTP (DIG-UTP) labelled probes ....................................................................................................... 30 2.1.4 Wax embedding and sectioning of WISH embryos ......................................31 2.2 C53 Full length cDNA Cloning............................................................................ 31 2.3 Generation of the C53 knockout mouse ..............................................................32 2.3.1 Isolation of genomic DNA using the Lambda FixII library ..........................32 2.3.2 Isolation of genomic DNA from BAG library (pBeloBACl 1 vector - Research Genetics) .................................................................................................32 2.3.3 Mapping of genomic DNA fragments ...........................................................33 2.3.4 Production of targeting vectors and targeting constucts ...............................33 2.3.5 General tissue culture techniques..................................................................35 2.3.6 Transfection and screening for targeted clones .............................................35 2.3.7 Blastocyst injection and breeding .................................................................37 2.3.8 Genotyping techniques ..................................................................................37 2.3.9 Reporter detection .........................................................................................38 2.4 In-Vitro differentiation of null ES cells ...............................................................39 2.4.1 Generation and in vitro differentiation of ES cell lines ................................39 2.4.2 cDNA synthesis and reverse transcription polymerase chain reaction (RT- PCR)...................................................................................................................... 39 2.5 Representational Difference Analysis (RDA) of RNA samples from C53 null yolk sac and liver............................................................................................................40 2.5.1 Isolation of RNA samples from embryonic yolk sac and liver and first strand cDNA synthesis ......................................................................................................40 2.5.2 Production of Difference Product (DP) 1 and 2............................................ 40 2.5.3 Cloning and sequencing of Difference Products ...........................................41 2.5.4 Confirmation of differential expression using quantitative PCR .................. 41 CHAPTER 3: SCREENING OF THE BEDDINGTON DISSECTED ENDODERM CDNA LIBRARY..................................................................... 44 3.1 Introduction .............................................................................................................44 3.1.1 The endoderm library screen ........................................................................ 44 3.2 Results...................................................................................................................... 46 3.2.1 Comparison of sequence from the endoderm library to sequence databases 46 3.2.2 Whole mount RNA in situ hybridisation of prioritised clones ......................46 3.3 Discussion ................................................................................................................. 51 3.3.1 The endoderm library screen for novel genes implicated in early patterning. ................................................................................................................................51 3.3.2 Expression o f‘restricted’ mRNAs from this study ....................................... 53 CHAPTER
Load more

Recommended publications
  • Smutty Alchemy
    University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2021-01-18 Smutty Alchemy Smith, Mallory E. Land Smith, M. E. L. (2021). Smutty Alchemy (Unpublished doctoral thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/113019 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Smutty Alchemy by Mallory E. Land Smith A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN ENGLISH CALGARY, ALBERTA JANUARY, 2021 © Mallory E. Land Smith 2021 MELS ii Abstract Sina Queyras, in the essay “Lyric Conceptualism: A Manifesto in Progress,” describes the Lyric Conceptualist as a poet capable of recognizing the effects of disparate movements and employing a variety of lyric, conceptual, and language poetry techniques to continue to innovate in poetry without dismissing the work of other schools of poetic thought. Queyras sees the lyric conceptualist as an artistic curator who collects, modifies, selects, synthesizes, and adapts, to create verse that is both conceptual and accessible, using relevant materials and techniques from the past and present. This dissertation responds to Queyras’s idea with a collection of original poems in the lyric conceptualist mode, supported by a critical exegesis of that work.
    [Show full text]
  • Suffrage Science Contents
    Suffrage science Contents Introduction Brenda Maddox and Vivienne Parry on sex and success in science 3 Love Professor Sarah-Jayne Blakemore and Dr Helen Fisher on love and social cognition 6 Life Professor Liz Robertson and Dr Sohaila Rastan on developmental biology and genetics 10 Structure Professor Dame Louise Johnson and Professor Janet Thornton on structural biology 15 Strife Professors Fiona Watt and Mary Collins on cancer and AIDS 19 Suffrage Heirloom Jewellery Designs to commemorate women in science 23 Suffrage Textiles Ribbons referencing the suffrage movement 33 Index of Featured Women Scientists Pioneering female contributions to Life Science 43 Acknowledgements Contributions and partnerships 47 Tracing Suffrage Heirlooms Follow the provenance of 13 pieces of Suffrage Heirloom Jewellery 48 1 A successful career in science is always demanding of intellect “ hard work and resilience; only more so for most women. ” Professor Dame Sally C Davies From top, left to right: (Row 1) Anne McLaren, Barbara McClintock, Beatrice Hahn, Mina Bissell, Brenda Maddox, Dorothy Hodgkin, (Row 2) Brigid Hogan, Christiane Nüsslein-Volhard, Fiona Watt, Gail Martin, Helen Fisher, Françoise Barré-Sinoussi, (Row 3) Hilde Mangold, Jane Goodall, Elizabeth Blackburn, Janet Thornton, Carol Greider, Rosalind Franklin, (Row 4) Kathleen Lonsdale, Liz Robertson, Louise Johnson, Mary Lyon, Mary Collins, Vivienne Parry, (Row 5) Uta Frith, Amanda Fisher, Linda Buck, Sara-Jayne Blakemore, Sohaila Rastan, Zena Werb 2 Introduction To commemorate 100 years of International Women’s Day in 2011, Suffrage Science unites the voices of leading female life scientists Brona McVittie talks to Vivienne Parry and Brenda Maddox about sex and success in science Dorothy Hodgkin remains the only British woman to Brenda Maddox is author of The Dark Lady of DNA, have been awarded a Nobel Prize for science.
    [Show full text]
  • Newsletter Australia and New Zealand Society for Cell and Developmental Biology INCORPORATED Winter 2012
    ANZSCDB Newsletter Australia and New Zealand Society for Cell and Developmental Biology INCORPORATED Winter 2012 Welcome to the mid provide a real chance for post year issue of the docs and students to strut their ANZSCDB newsletter. stuff and get noticed locally. It has already been a We are very happy to support busy year of activities these initiatives, a process I and meetings so far witnessed first hand at the NSW for the society, and meeting in March this year. The we round up some increased funding for the yearly of the activities that state meetings has allowed the have occurred and invitation of interstate speakers, highlight upcoming which has proved a bit of a draw meetings and society card for these meetings. In related events. In this issue we the next few weeks we will be take some time to celebrate one calling for new representatives. of the senior members of the Each year one of the state/ ANZSCDB community, Patrick NZ representatives steps Tam. Patrick has reached the down and we are now calling age where the combination of for nominations for keen and experience and wisdom peak, engaged new representatives, and it is only right and fitting in particular from the ACT that we acknowledge the and from Tasmania (who are important contributions Patrick currently not represented). Read Up On: has made to our discipline This opportunity is an excellent throughout his career. We way to engage with your President's Report preview COMBIO in Adelaide and peers, to advance and promote celebrate the awarding of the wider interest in our field society’s top awards for 2012.
    [Show full text]
  • Shooting the Messenger Genetics Group Targets Disease Markers in the Human Sequence Physicists Show What Really Matters Europe H
    12 July 2001 Nature 412: 6843 (2001) © Macmillan Publishers Ltd. Shooting the messenger 103 The Intergovernmental Panel on Climate Change has a creditable record of developing a scientific consensus and delivering it to policy-makers. What its critics really object to are the facts. Genetics group targets disease markers in the human 105 sequence Physicists show what really matters 105 Europe hooks up with China for space first 106 Institutes prepare for pioneering bioinformatics work 106 Stem-cell fudge finds no favour with biologists 107 Bush plots raid on NIH funds to finance AIDS initiative 107 Royal Society disputes value of carbon sinks 108 Battle to save beleaguered beluga 108 UN backs transgenic crops for poorer nations 109 Arctic university gives collaboration pole position 109 news in brief 110 Consensus science, or consensus politics? 112 To some, the Intergovernmental Panel on Climate Change represents the pinnacle of scientific collaboration. To others, it is a victory for politics over science. Mark Schrope talks to the experts debating our planet's future. Alien versus predator 115 Can invasive species be controlled by introducing their natural enemies? The idea has a chequered history. But as safety testing improves, it is now gaining currency. Jonathan Knight reports. Seeking, sometimes finding, that elusive chemistry 117 Despite all the discipline's achievements, opinion is divided as to whether chemistry is getting the recognition it deserves — and needs — in order to keep attracting new talent. Researchers are popular,
    [Show full text]
  • MRC Investigators and Directors Directory of Research Programmes at MRC Institutes and Units Foreword
    SUMMER 2021 MRC Investigators and Directors Directory of research programmes at MRC Institutes and Units Foreword I am delighted to introduce you to the exceptional To support the MRC Investigators and Directors researchers at our MRC Institutes and Units – the in advancing medical research, MRC provides MRC Investigators and their Directors. core funding to the MRC Institutes and University Units where they carry out their work. These In November 2020, MRC established the new title establishments cover a huge breadth of medical of “MRC Investigator” for Programme Leaders (PL) research from molecular biology to public health. and Programme Leader Track (PLT) researchers at As you will see from the directory, the MRC MRC Institutes and Units. These individuals are Investigators and Directors are making considerable world-class scientists who are either strong leaders advances in their respective fields through their in their field already (PLs) or are making great innovative and exciting research programmes. Their strides towards that goal (PLTs). Based on what accomplishments have been recognised beyond they have achieved in their research careers so far, MRC and many have been awarded notable prizes the title will no doubt become synonymous with and elected to learned societies and organisations. scientific accomplishment, impact and integrity. As well as being widely recognised within the MRC endeavours to do everything it can to support scientific and academic communities, the well- its researchers at all career stages. For this reason, established and newer title of “Director” and we chose not to distinguish between levels of “MRC Investigator”, respectively, are a signal seniority within the new title.
    [Show full text]
  • Back Matter (PDF)
    Your time is better spent researching science - we'll research the supplies. Manipulating the Mouse Embryo A Laboratory Manual, Second Edition By Brigid Hogan, Vanderbilt University Medical School; Rosa Beddington, National Institute for Medical Research, London; Frank Costantini, Columbia University; Elizabeth Lacy, Memorial Sloan-Kettering Cancer Center " -Here's what the reviewers have to say: "Anyone embarking on a research career in mam- malian developmental biology would be well advised to regard this book as the Bible of embryo manipula- tion techniques. Rather like Kellogg's Corn Flakes, this book is often copied but rarely surpassed; still the original and the best. Almost every method that might be required by a budding embryologist is cov- ered comprehensively and clearly .... For people com- pletely new to mouse embryology there is also an ex- cellent chapter summarizing early mouse develop- ment with colour figures to help visualize conceptual- ly difficult events like gastrulation and turning. Several areas have been expanded, including the sec- tion summarizing mouse development, analysis of transgenic mice, and visualization of transgene and endogenous gene expression. A glossary of the Section C. Recovery, Culture, and Transfer of Embryos mouse genome is included for the first time .... The and Germ Cells second edition is significantly different from the first Section D. In Vitro Manipulation of Preimplantation and contains new methods and extended information Embryos that fully justifies spending the academic's meagre Section E. Production of Transgenic Mice pay rise to buy the book. Section F. Isolation, Culture, and Manipulation of Embryonic Stem Cells In conclusion, this methods manual is highly recom- Section G.
    [Show full text]
  • Myelnikov Phd Ethos
    Transforming Mice: Technique and communication in the making of transgenic animals, 1974–1988 Dmitriy Myelnikov Department of History and Philosophy of Science Emmanuel College University of Cambridge September 2014 Corrected version, May 2015 This dissertation is submitted for the degree of Doctor of Philosophy i This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where spe- cifically indicated in the text. No parts of this dissertation have been submitted for other qualifications. It does not exceed 80,000 words. Dmitriy Myelnikov ii Table of Contents Acknowledgements iii Abbreviations v Introduction 1 Note on sources 14 Chapter 1. The rise of the mouse embryo, 1941–1970 17 §1. The genetic mammal 19 §2. Standards and embryos 24 §3. Manipulating development 32 §4. Molecular promises 41 Conclusion 50 Chapter 2. Recombinant networks: The moral economy of genetic engineering in the 1970s 52 §1. Gene transfer and somatic cell genetics 54 §2. Viruses and embryos: The Jaenisch-Mintz collaboration 61 §3. Recombinant exchanges 67 §4. “DNA-mediated gene transfer”: Expanding the networks 75 Conclusion 81 Chapter 3. Putting genes into mice: Promises, experimental trajectories and expertise 83 §1. “Possibilities and realities” 85 §2. Research agendas 95 §3. Mastering microinjection 105 §4. Recruiting molecular expertise 110 Conclusion 116 Chapter 4. Negotiating new mice: News, journals and priority 118 §1. Breaking the news 122 §2. “A one-way trip to the Brave New World”? 125 §3. Journals and the politics of priority 133 §4. Criteria of success 144 i §5. “These mice, that we call transgenic” 151 Conclusion 156 Chapter 5.
    [Show full text]
  • 2000 to 2010 Lister Annual Report and Accounts
    t h e l is t e r in stitu te o f p r e v e n t iv e m ed ic in e The White House, 70 High Road, Bushey Heath, Hertfordshire WD23 IGG REPORT AND FINANCIAL STATEMENTS for the year ended 31 December The Lister Institute of Preventive Medicine is a company limited by guarantee (England 34479) and a registered charity (206271) Errata Page 5 The reference to the Statement of Financial Activities at the foot of the page should be to page 19 (not 18). Page 13 The reference to Note I in the first paragraph should be to page 21 (not 20). Pages 19 and 20 Reference is to be made to the Notes on pages 21 to 24 (not 20 to 24). The cover portrait of Lord Lister is reproduced by courtesy of the Royal Veterinary College THE LISTER INSTITUTE OF PREVENTIVE MEDICINE THE GOVERNING BODY for the year ended 31 December 2001 Dr Anne L McLaren, DBE, MA, DPhil, FRCOG, FRS, Choir Peter W Allen, MA, FCA, ClMgt, Hon Treasurer W Lawrence Banks, CBE G James M Buckley Professor H John Evans, CBE, PhD, FRCPE, FRSE C Edward Guinness, CVO (until June 2001) Hon Rory M B Guinness Professor Sir Alec J Jeffreys, DPhil, FRS Dr AlanJ Munro, PhD Professor Richard N Perham, ScD, FRS Professor J G Patrick Sissons, MD, FRCP, FRCPath Professor Anne E Warner, PhD, FRS Secretary, and Clerk to the Governors: F KCowey, MA, BSc, DAS Address of principal office of the charity: The White House 70 High Road Bushey Heath Hertfordshire WD23 IGG Professional advisors Bankers Investment Advisors Messrs Coutts & Co ) P Morgan Fleming Asset Management Ltd St Martins Office Finsbury Dials 440 Strand 20 Finsbury Street London WC2R OQS London EC2Y 9AQ Solicitors Auditors Macfarlanes PricewaterhouseCoopers 10 Norwich Street I Embankment Place London EC4A IBD London WC2N 6HR THE LISTER INSTITUTE OF PREVENTIVE MEDICINE FINANCIAL REPORT OF THE GOVERNING BODY for the year ended 3 I December 2001 The Institute is a company limited by guarantee Future operations and has charitable status.
    [Show full text]
  • Michaelmas 2019
    fusionTHE NEWSLETTER OF THE SIR WILLIAM DUNN SCHOOL OF PATHOLOGY ISSUE 18 . MICHAELMAS 2019 Focus on Structural Biology Spotlight on Interview with Spin-Outs Richard Gardner Cell Fate Decisions in the Early Embryo A Brief History of Macrophages 2 / FUSION . MICHAELMAS 2019 Welcome In the last edition of Fusion, I wrote about the celebration of penicillin we held when we were response to viral infection and will be an awarded a national blue plaque for the Dunn School building. This time I want to focus on the excellent fit with many of our other future. Changes are afoot in Oxford science and I believe these will place the Dunn School in an groups. even stronger position globally. Important science is increasingly interdisciplinary, and I have been Among many achievements by members involved in long discussions about how Oxford should respond to this evolution, moving away from of the Dunn School this year, I would like narrowly-defined subject areas. Two new initiatives promise to define a new ethos for Oxford to single out the award of two research: the establishment of a new institute and of a brand new college. international prizes to Tanmay Bharat, one of our early career group leaders (see opposite). Tanmay was the recipient of Science Library at the end of South Parks Road. the Sir John Kendrew Award from the The relevant message here is that, like the European Molecular Biology Laboratory, as Dorothy Hodgkin Centre, Parks College reflects well as being the only non-US recipient of the new Oxford philosophy of removing all a Vallee Scholar Award, recognising both boundaries between disciplines.
    [Show full text]
  • BSDB Newsletter
    Contents BSDB Newsletter Summer !""# Volllume !$%%% Number & Ediiitoriiialll Contents This issue of the Newsletter sees the introduction of Ediiitoriiialll & Contents & several changes to the standard BSDB format, not the least of which is a new adventure for the Autumn From the Chaiiirman ! Meeting. Following an initiative from our Chairman, Waddiiington Medalll ! Alfonso Martinez-Arias has teamed up with col- leagues in the French Society for Developmental Biol- News (& Viiiews) # ogy to organise a late summer scorcher on the Cote Committee changes d’Azur. With BA aggressively competing with EasyJet New ‘Open Access’ Journal and Ryanair, you now have no excuse for turning up Greenfield Report on Women in Science pale and washed out for the start of the next academic The future of NIMR year… RS under scrutiny Big changes too on the website. Thanks to a great ef- March of Dimes Prize for Developmental Biology fort by Kate Storey, our new Website Co-ordinator, the From the Treasurer ( BSDB website now looks better than ever. More im- Travel Grants portantly it also functions better than ever. Aside from Financial Statement 2002 the listings of committee members and meetings that it Graduate Students ) * + has always had, you can now download all the forms that have traditionally been found in the back of the BSDB Autumn Meetiiing , Newsletter (Indeed, I would like to drop these from the Future BSDB Meetiiings - next issue, so let me know soon if this is a bad idea). Kate has also substantially increased the range of in- Other Relllated Meetiiings & Courses &" formation and links to related sites that are available.
    [Show full text]