Methods in Molecular Biology TM Methods in Molecular Biology TM HUMANA PRESS HUMANA PRESS Edited by Marten H. Hofker Jan van Deursen Transgenic Mouse Methods and Protocols VOLUME 209 Edited by Marten H. Hofker Jan van Deursen Transgenic Mouse Methods and Protocols Transgenic Mouse Methods and Protocols M E T H O D S I N M O L E C U L A R B I O L O G Y TM John M. Walker, S ERIES E DITOR 217. Neurogenetics: Methods and Protocols, edited by Nicholas T. Potter, 2003 216. PCR Detection of Microbial Pathogens: Methods and Pro- tocols, edited by Konrad Sachse and Joachim Frey, 2003 215. Cytokines and Colony Stimulating Factors: Methods and Protocols, edited by Dieter Körholz and Wieland Kiess, 2003 214. Superantigen Protocols, edited by Teresa Krakauer, 2003 213. Capillary Electrophoresis of Carbohydrates, edited by Pierre Thibault and Susumu Honda, 2003 212. Single Nucleotide Polymorphisms: Methods and Protocols, edited by Piu-Yan Kwok, 2003 211. Protein Sequencing Protocols, 2nd ed., edited by Bryan John Smith, 2003 210. MHC Protocols, edited by Stephen H. Powis and Robert W. Vaughan, 2003 209. Transgenic Mouse Methods and Protocols, edited by Marten Hofker and Jan van Deursen, 2002 208. Peptide Nucleic Acids: Methods and Protocols, edited by Peter E. Nielsen, 2002 207. Recombinant Antibodies for Cancer Therapy: Methods and Protocols. edited by Martin Welschof and Jürgen Krauss, 2002 206. Endothelin Protocols, edited by Janet J. Maguire and Anthony P. Davenport, 2002 205. E. coli Gene Expression Protocols, edited by Peter E. Vaillancourt, 2002 204. Molecular Cytogenetics: Protocols and Applications, edited by Yao-Shan Fan, 2002 203. In Situ Detection of DNA Damage: Methods and Protocols, edited by Vladimir V. Didenko, 2002 202. Thyroid Hormone Receptors: Methods and Protocols, edited by Aria Baniahmad, 2002 201. Combinatorial Library Methods and Protocols, edited by Lisa B. English, 2002 200. DNA Methylation Protocols, edited by Ken I. Mills and Bernie H, Ramsahoye, 2002 199. Liposome Methods and Protocols, edited by Subhash C. Basu and Manju Basu, 2002 198. Neural Stem Cells: Methods and Protocols, edited by Tanja Zigova, Juan R. Sanchez-Ramos, and Paul R. Sanberg, 2002 197. Mitochondrial DNA: Methods and Protocols, edited by William C. Copeland, 2002 196. Oxidants and Antioxidants: Ultrastructural and Molecular Biology Protocols, edited by Donald Armstrong, 2002 195. Quantitative Trait Loci: Methods and Protocols, edited by Nicola J. Camp and Angela Cox, 2002 194. Posttranslational Modifications of Proteins: Tools for Functional Proteomics, edited by Christoph Kannicht, 2002 193. RT-PCR Protocols, edited by Joe O’Connell, 2002 192. PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen and Harry W. Janes, 2002 191. Telomeres and Telomerase: Methods and Protocols, edited by John A. Double and Michael J. Thompson, 2002 190. High Throughput Screening: Methods and Protocols, edited by William P. Janzen, 2002 189. GTPase Protocols: The RAS Superfamily, edited by Edward J. Manser and Thomas Leung, 2002 188. Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 187. PCR Mutation Detection Protocols, edited by Bimal D. M. Theophilus and Ralph Rapley, 2002 186. Oxidative Stress Biomarkers and Antioxidant Protocols, ed- ited by Donald Armstrong, 2002 185. Embryonic Stem Cells: Methods and Protocols, edited by Kursad Turksen, 2002 184. Biostatistical Methods, edited by Stephen W. Looney, 2002 183. Green Fluorescent Protein: Applications and Protocols, edited by Barry W. Hicks, 2002 182. In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Braman, 2002 181. Genomic Imprinting: Methods and Protocols, edited by Andrew Ward, 2002 180. Transgenesis Techniques, 2nd ed.: Principles and Protocols, edited by Alan R. Clarke, 2002 179. Gene Probes: Principles and Protocols, edited by Marilena Aquino de Muro and Ralph Rapley, 2002 178. Antibody Phage Display: Methods and Protocols, edited by Philippa M. O’Brien and Robert Aitken, 2001 177. Two-Hybrid Systems: Methods and Protocols, edited by Paul N. MacDonald, 2001 176. Steroid Receptor Methods: Protocols and Assays, edited by Benjamin A. Lieberman, 2001 175. Genomics Protocols, edited by Michael P. Starkey and Ramnath Elaswarapu, 2001 174. Epstein-Barr Virus Protocols, edited by Joanna B. Wilson and Gerhard H. W. May, 2001 173. Calcium-Binding Protein Protocols, Volume 2: Methods and Techniques, edited by Hans J. Vogel, 2001 172. Calcium-Binding Protein Protocols, Volume 1: Reviews and Case Histories, edited by Hans J. Vogel, 2001 171. Proteoglycan Protocols, edited by Renato V. Iozzo, 2001 170. DNA Arrays: Methods and Protocols, edited by Jang B. Rampal, 2001 169. Neurotrophin Protocols, edited by Robert A. Rush, 2001 168. Protein Structure, Stability, and Folding, edited by Kenneth P. Murphy, 2001 167. DNA Sequencing Protocols, Second Edition, edited by Colin A. Graham and Alison J. M. Hill, 2001 166. Immunotoxin Methods and Protocols, edited by Walter A. Hall, 2001 165. SV40 Protocols, edited by Leda Raptis, 2001 164. Kinesin Protocols, edited by Isabelle Vernos, 2001 163. Capillary Electrophoresis of Nucleic Acids, Volume 2: Practical Applications of Capillary Electrophoresis, edited by Keith R. Mitchelson and Jing Cheng, 2001 162. Capillary Electrophoresis of Nucleic Acids, Volume 1: Introduction to the Capillary Electrophoresis of Nucleic Acids, edited by Keith R. Mitchelson and Jing Cheng, 2001 161. Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001 160. Nuclease Methods and Protocols, edited by Catherine H. Schein, 2001 159. Amino Acid Analysis Protocols, edited by Catherine Cooper, Nicole Packer, and Keith Williams, 2001 158. Gene Knockoout Protocols, edited by Martin J. Tymms and Ismail Kola, 2001 Transgenic Mouse Methods and Protocols Edited by Marten H. Hofker Department of Molecular Genetics University of Maastricht Maastricht, The Netherlands Jan van Deursen Department of Pediatrics and Adolescent Medicine Mayo Clinic, Rochester, MN Humana Press Totowa, New Jersey M E T H O D S I N M O L E C U L A R B I O L O G Y TM © 2003 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology™ is a trademark of The Humana Press Inc. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Transgenic mouse methods and protocols / edited by Marten H. Hofker, Jan van Deursen. p. cm. (Methods in molecular biology ; 209) Includes bibliographical references and index. ISBN 0-89603-915-3 (alk. paper) 1. Transgenic mice Laboratory manuals. I. Hofker, Marten H. II. Deursen, Jan van, 1930– III. Methods in molecular biology (Totowa, N.J.) ; v. 209 QH442.6 .T724 2002 616'.93 dc21 2002068581 Preface v Mice play an important role in biomedical research. Genetically, the mouse is very similar to the human species, and most human genes are conserved in the mouse. Moreover, the arrangement of genes on chromosomes is similar in humans and mice. Mouse models have been established for a wide variety of human disorders including cancer, cardiovascular diseases, and behavioral disorders. Transgenic Mouse Methods and Protocols covers the production and analysis of transgenic and knockout mice. The book also includes a review of mouse models for a wide range of genetic aberrations associated with human disease. Many researchers played a crucial role in developing mouse technology to the excellent state of art that has now been achieved. Landmarks include the generation of (i) transgenic mice, (ii) pluripotent embryonic stem (ES) cell cultures, (iii) gene knockout mice, (iv) tissue-specific knockouts, and (v) systems for inducible gene expression mice. Most of these landmarks have not been achieved in other mammalian systems with a comparable efficiency. In part, this is attributable to the availability of hundreds of different inbred mouse strains, which allowed researchers to choose from a wide range of strains while establishing these technologies. Transgenic Mouse Methods and Protocols has essentially the same format as previous volumes of the series Methods in Molecular Biology. Since mouse technology offers a wide range of possibilities, most chapters will provide the rationale for choosing the given protocol, which is then described in step-by-step detail. In addition, two chapters will describe alternative strategies for studying gene function in the mouse, i.e., random mutagenesis and viral gene transduction. The book can be roughly divided into three topics: a general introduction describing how to deal with mice and how to generate transgenics; a technology part where new methods will be presented, including conditional and induced knockout and transgenic mice; and the final chapters, which highlight working with mice in several major research areas, including cancer, immunology, and atherosclerosis. We would like to thank the authors for their excellent contributions and Wilma Claessen for editorial assistance. We are very grateful to Be Wieringa, Anton Berns, and Robin Lovell Badge for leading us into the world of gene-targeting and ES cell technology. Marten Hofker Jan van Deursen Preface v Contributors ix Color Plates xiii 1 Introduction: The Use of Transgenic Mice in Biomedical Research Marten H. Hofker 1 2 Genetic Modification of the Mouse: General Technology; Pronuclear and Blastocyst Injection J. Willem Voncken 9 3 In Vitro Fertilization/Cryopreservation Susan Marschall 0and Martin Hrabe de Angelis 35 4 Transgene Design J. Willem Voncken 51 5 Generating Conditional Mouse Mutants via Tetracycline-Controlled Gene Expression Kai Schönig and Hermann Bujard 69 6 Manipulating Large Insert Clones for Transgenesis Sally P. A. McCormick, Catherine Y. Y. Liu, Stephen G. Young, and Lars B. Nielsen 105 7 Reporter Molecules in Genetically Engineered Mice Thomas L. Saunders 125 8 Gene Targeting in Mouse Embryonic Stem Cells Jan van Deursen 145 9 Conditional Knockout Mice Ralf Kühn and Frieder Schwenk 159 10 Knockin Approaches Anton J. M. Roebroek, Xiaosheng Wu, and Richard J. Bram 187 11 Aggregation of Embryos and Embryonic Stem Cells Ronald Maatman, Marina Gertsenstein, Emile de Meijer, Andras Nagy, and Kristina Vintersten 201 12 Andenovirus-Mediated Gene Transfer Ko Willems van Dijk, Kyriakos E. Kypreos, Christine d’Oliveira, and Frits J. Fallaux 231 vii Contents ˆ viii Contents 13 Random ENU Mutagenesis Dian Soewarto, Véronique Blanquet, and Martin Hrabe de Angelis 249 14 Autopsy and Histologic Analysis of the Transgenic Mouse Marion J. J. Gijbels and Menno P. J. de Winther 267 15 Bone Marrow Transplantations to Study Gene Function in Hematopoietic Cells Menno P. J. de Winther and Peter Heeringa 281 16 Quantification of Atherosclerosis in Mice Alan Daugherty and Stewart C. Whitman 293 17 Genetically Modified Mice in Cancer Research David A. Largaespada 311 18 Neuropathobiology in Transgenic Mice: The Case of Alzheimer’s Disease Jo Van Dorpe, Kurt Spittaels, Chris Van den Haute, Ilse Dewachter, Dieder Moechars, Hugo Geerts, and Fred Van Leuven 333 Index 363 ˆ ix Contributors VÉRONIQUE BLANQUET • GSF Research Centre for Environment and Health, Institute of Experimental Genetics, Neuherberg, Germany R ICHARD J. BRAM • Department of Pediatrics and Adolescent Medicine, Mayo Clinic, Rochester, MN H ERMANN BUJARD • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany A LAN DAUGHERTY • Gill Heart Institute, Division of Cardiovascular Medicine, University of Kentucky, Lexington KY J AN VAN DEURSEN • Department of Pediatrics and Adolescent Medicine, Mayo Clinic, Rochester, MN I LSE DEWACHTER • Experimental Genetics Group, Center for Human Genetics, Flemish Institute for Biotechnology, Katholieke Universiteit Leuven, Leuven, Belgium K O WILLEMS VAN DIJK • Departments of General Internal Medicine and of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands J O VAN DORPE • Experimental Genetics Group, Center for Human Genetics, Flemish Institute for Biotechnology, Katholieke Universiteit Leuven, Leuven, Belgium F RITS J. FALLAUX • Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands H UGO GEERTS • Janssen Research Foundation, Beerse, Belgium M ARINA GERTSENSTEIN • Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada M ARION J. J. GIJBELS • Department of Molecular Genetics, Universiteit Maastricht, Maastricht, The Netherlands C HRIS VAN DEN HAUTE • Experimental Genetics Group, Center for Human Genetics, Flemish Institute for Biotechnology, Katholieke Universiteit Leuven, Leuven, Belgium P ETER HEERINGA • Department of Clinical and Experimental Immunology, Universiteit Maastricht, Maastricht, The Netherlands M ARTEN H. HOFKER • Department of Molecular Genetics, Universiteit Maastricht, Maastricht, The Netherlands M ARTIN HRABE DE ANGELIS • GSF Research Centre for Environment and Health, Institute of Experimental Genetics, Neuherberg, Germany R ALF KÜHN • Artemis Pharmaceuticals GmbH, Cologne, Germany ˆ [...]... Husbandry: Choice of Genetic Background As transgenic technology and protocols are described in this chapter, it is assumed that the researcher has access to a professional and well-equipped laboratory animal facility with ample experience in mouse handling, breeding, and the surgical and/ or dissection techniques required for the production of transgenic mice Animals used to generate transgenic and. .. evidence may come from human genetic studies Transgenic mice are generated to confirm the role of a disease mutation and will help to unravel the underlying molecular and biochemical mechanisms In addition, the disease model will help in designing novel From: Methods in Molecular Biology, vol 209: Transgenic Mouse Methods and Protocols Edited by: M H Hofker and J van Deursen © Humana Press Inc., Totowa,... targeting Hence the mouse became the species of choice for biomedical research At present, it is feasible to generate both loss-of-function and gain-of-function models and also to restrict these changes to specific cell types and developmental stages (discussed below) 4 Contents of This Book 4.1 Basic Mouse Technology Chapter 2 is devoted to general mouse technology, including mouse husbandry and microinjections... embryos after birth (see Subheadings 3.1 and 3.1.2.) Procedures and requirements concerning animals used for fertilized oocyte production and for oviduct transfer and guidelines for equipment and microsurgical techniques are described in excellent detail in Manipulating the Mouse Embryo by Hogan et al (1) and in the video guide Transgenic Techniques in Mice by Pedersen and Rossant (2) In addition, these... injection and uterine transfer are discussed in this chapter as well (see Fig 1 and Subheadings 3.2., 3.3., and 3.4.) For basic molecular cloning techniques and strategies and molecular detection methods such as polymerase chain reaction (PCR)-based or Southern blot analysis, we recommend Molecular Cloning A Laboratory Manual by Sambrook, et al (6) Transgenic construct design is discussed in Chapter 4 1.1 Mouse. .. a particular mouse model The Jackson Laboratory can provide a large number of different mouse models (see http://www.jax.org) that emerged from transgenic studies and were obtained after random mutation events (either spontaneous or induced) Many other transgenic studies are initiated on the basis of the predicted gene function Such predictions come from homology between the mouse gene and genes in... microinjection of DNA directly into one of the pronuclei of a fertilized mouse egg (1–5) Typically, microinjected DNA will integrate at one site within the genome, often as a concatamer (a multicopy insertion), arranged in a head-to-tail fashion From: Methods in Molecular Biology, vol 209: Transgenic Mouse Methods and Protocols Edited by: M H Hofker and J van Deursen © Humana Press Inc., Totowa, NJ 9 10 Voncken... 100-fold The main strength of the mouse models is that at the molecular and cellular levels, they correspond well to the human However, one should not expect the mouse to copy human disease completely 3 Transgenic Technology: “Gain of Function” Versus “Loss of Function” Since the publication of one of the landmark papers on “conventional transgenics” in 1982 by Palmiter and colleagues (2), the technology... Thomas, K R., and Capecchi, M R (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes Nature 336, 348–352 4 Somia, N and Verma, I M (2000) Gene therapy: trials and tribulations Nat Rev Genet 1, 91–99 Genetic Modification of the Mouse 9 2 Genetic Modification of the Mouse General Technology; Pronuclear and Blastocyst... abdomen with 70% ethanol, and make an incision over the xiphoid (Fig 2) 5 Grasp the skin with both hands and pull back firmly in opposite directions (rostrally and caudally), essentially skinning the mouse completely (Fig 2) 6 Make incisions in the body wall by lifting it with tweezers, and cutting it with scissors; expose the body cavity completely 7 Move intestines aside gently and grab ovaries by the . Deursen Transgenic Mouse Methods and Protocols Transgenic Mouse Methods and Protocols M E T H O D S I N M O L E C U L A R B I O L O G Y TM John M. Walker, S ERIES E DITOR 217. Neurogenetics: Methods. cardiovascular diseases, and behavioral disorders. Transgenic Mouse Methods and Protocols covers the production and analysis of transgenic and knockout mice. The book also includes a review of mouse models for. Damage: Methods and Protocols, edited by Vladimir V. Didenko, 2002 202. Thyroid Hormone Receptors: Methods and Protocols, edited by Aria Baniahmad, 2002 201. Combinatorial Library Methods and Protocols,