Gene Knockout Protocols

433 Pages·2001·4.239 MB·English

by  Paul J. Hertzog, Ismail Kola (auth.), Martin J. Tymms, Ismail Kola (eds.)

Gene Knockout Protocols
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M E T H O DS IN M O L E C U L AR B I O L O G Y1
Gene Knockout
Protocols
Edited by
Martin J. Tymms
National Vision Research Institute of Australia,
Carl ton, Victoria, Australia
and
Ismail Kola
Centre for Functional Genomics and Human Disease,
Institute of Reproduction and Development, Monash University,
Clayton, Victoria, Australia
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Library of Congress Cataloging-in-Publication Data
Gene knockout protocols / edited by Martin J. Tymms and Ismail Kola.
p.cm. --(Methods in molecular biology ; 158)
Includes bibliographical references and index.
ISBN 0-89603-572-7 (alk. paper)
1. Gene targeting-Laboratory manuals. 2. Transgenic mice-Laboratory manuals. 1.
Tymms, Martin J. II. Kola, Ismail. III. Methods in molecular biology (Clifton, N.J.) ; v.
158.
QH442.3 .G453 2000
572.8'77-dc21
Preface
As the major task of sequencing the human genome is near completion
and full complement of human genes are catalogued, attention will be focused
on the ultimate goal: to understand the normal biological functions of these
genes, and how alterations lead to disease states. In this task there is a severe
limitation in working with human material, but the mouse has been adopted as
the favored animal model because of the available genetic resources and the
highly conserved gene conservation linkage organization.
In just of ten years since the first gene-targeting experiments were pre-
formed in embryonic stem (ES) cells and mutations transmitted through the
mouse germline, more than a thousand mouse strains have been created. These
achievements have been made possible by pioneering work that showed that
ES cells derived from preimplantation mouse embryos could be cultured for
prolonged periods without differentiation in culture, and that homologous recom-
bination between targeting constructs and endogenous DNA occurred at a fre-
quency sufficient for recombinants to be isolated. In the next few years the
mouse genome will be systematically altered, and the techniques for achiev-
ing manipulations are constantly being streamlined and improved.
Recently new technologies have developed for inducible gene expres-
sion in transgenic mice that in combination with conventional gene targeting
can give temporal and tissue-specific expression. These advances have been
spurred on by the desire to study the function of genes that show an embry-
onic-lethal phenotype when deleted or "knocked out". Gene targeting in mice
initially concentrated on making gene knockouts or null mutations, but increas-
ingly the technology is being used to create subtle point mutations to simulate
human disease states.
Gene Knockout Protocols brings together distinguished contributors with
extensive experience in the gene targeting and mouse genetics fields. In line
with the successful format of Methods in Molecular Biology, the volume con-
tains step-by-step protocols for the design of targeting constructs to protocols
for the analysis of the mouse phenotype. Emphasis has been paid to the inclu-
sion of other techniques used in mouse genetics that are relevant to research-
ers performing gene targeting. These include embryo transplantation, In vitro
vi Preface
ES cell differentiation, creation of aggregation chimeras, mouse pathology, embryo
cryopreservation, and transplantation. Issues such as the use of existing mouse
mutation resources and the influence of genetic background and epigenetic
effects upon phenotype are also covered. We hope that Gene Knockout Proto-
cols will be an invaluable source of proven protocols for those just entering
the field of gene targeting, but also a valuable reference for researchers in the
process of describing the phenotype of mutant mice.
Martin J. Tymms
Ismail Kola
Contents
Preface v
Contributors ix
1 Overview: Gene Knockouts
Paul J. Hertzog and Ismail Kola 1
2 Isolation and Maintenance of Primate Embryonic Stem Cells
Vivienne S. Marshall, Michelle A. Waknitz,
and James A. Thomson 11
3 Gene Targeting in ES Cells
Thomas M, DeChiara 19
4 Manipulating Mouse Embryonic Stem Cells
Lino Tessarollo 47
5 Gene Targeting in a Centralized Facility
Louise D. Barnett and Frank Kontgen ,.65
6 The LoxP/CRE System and Genome Modification
Trevor J. Wilson and Ismail Kola 83
7 Creation and Use of a Cre Recombinase Transgenic Database
Andras Nagy and Lynn Mar 95
8 Choice of Mouse Strains for Gene Targeting
Mireille H. Lahoud, Trevor J. Wilson, and Paul J. Hertzog 107
9 Isolation, Microinjection, and Transfer of Mouse Blastocysts
Angelika Bonin, Susan W. Reid, and Lino Tessarollo 121
10 Aggregation Chimeras: Combining ES Cells, Diploid
and Tetraploid Embryos
Mika Tanaka, Anna-Katerina Hadjantonakis,
and Andras Nagy 135
11 How to Study Pathologic Phenotypes of Knockout Mice
Roderick T. Bronson 155
12 Analysis of Hematopoietic Phenotypes
in Knockout Mouse Models
Ivan Bertoncello and Brenda Williams , 181
vii
viii Contents
13 Isolation of Embryonic Fibroblasts and Their Use in the In Vitro
Characterization of Gene Function
Paul J. Hertzog 205
14 Influence of Genetic Background on Knockout Mouse Phenotypes
L. Philip Sanford, Suhas Kallapur, llona Ormsby,
and Thomas Doetschman 217
15 Lineage Allocation During Early Embryogenesis: Mapping
of the Neural Primordia and Application to the Analysis
of Mouse Mutants
Gabriel A. Quintan, Bruce P. Davidson, and Patrick P. L. Tarn... 227
16 Generation of Double-Knockout Embryonic Stem Cells
Hein te Riele, Conny Brouwers, and Marleen Dekker 251
17 In Vitro Differentiation of Embryonic Stem Cells and Analysis
of Cellular Phenotypes
Anna M. Wobus, Kaomei Guan, and Uta Pich 263
18 Embryonic Stem Cells in the Study of Hematopoiesis
Francesca Lazner, Ismail Kola, Elizabeth Stadler,
and Andrew G. Elefanty 287
19 Interferon-lnducible ES Cell Expression Systems
Linda M. Whyatt and Peter D. Rathjen 301
20 Transgenic Studies in the Mouse: Improving the Technology
Towards a Conditional Temporal and Spatial Approach
Sika Ristevski 319
21 In Vivo Libraries of Large Insert Transgenic Mice
for Genetic Mapping
Desmond J. Smith 335
22 Epigenetic Effects on Transgene Expression
Emma Whiteiaw, Heidi Sutherland, Margot Kearns, Hugh Morgan,
Linda Weaving, and David Garrick 351
23 Positional-Candidate Cloning of Genes from Mouse Mutants
Karen B. Avraham 369
24 Genetically Engineered Mice: Husbandry and Resources
John J. Sharp, Carol C. Linder, and Larry E. Mobraaten 381
25 Embryo Cryopreservation for Transgenic Mouse Lines
Jillian M. Shaw and Magosaburo Kasai 397
Index 421
Contributors
KAREN B. AVRAHAM • Department of Human Genetics and Molecular
Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
LOUISE D. BARNETT • The Walter and Eliza Hall Institute of Medical Research,
Melbourne, Australia
IVAN BERTONCELLO • Trescowthick Research Laboratories, Peter MacCallum
Cancer Institute, Melbourne, Australia
ANGELIKA BONIN • BASF Bioresearch Corporation, Worcester, MA.
RODERICK T. BRONSON • Human Nutrition Research Center, Boston, MA
CONNY BROUWERS • Division of Molecular Carcinogenesis, The Netherlands
Cancer Institute, Amsterdam, The Netherlands
BRUCE P. DAVIDSON • Embryology Unit, Children's Medical Research
Institute, Sydney, Australia
THOMAS M. DECHIARA • Regeneron Pharmaceuticals, Tarrytown, NY
MARLEEN DEKKER • Division of Molecular Carcinogenesis, The Netherlands
Cancer Institute, Amsterdam, The Netherlands
THOMAS DOETSCHMAN • Department of Molecular Genetics, Biochemistry
and Microbiology, University of Cincinnati College of Medicine,
Cincinnati, OH
ANDREW G. ELEFANTY • The Walter and Eliza Hall Institute of Medical
Research, Melbourne, Australia
DAVID GARRICK. • Department of Biochemistry, University of Sydney,
Sydney, Australia
KAOMEI GUAN • In Vitro Differentiation Group, IPK Gatersleben,
Gatersleben, Germany
ANNA-KATERINA HADJANTONAKIS • Samuel Lunenfeld Research Institute,
Mount Sinai Hospital, Toronto, Ontario, Canada
PAUL J. HERTZOG • Centre for Functional Genomics and Human Disease,
Monash University, Melbourne, Australia
SUHAS KALLAPUR • Department of Neonatology, Children's Hospital,
Cincinnati, OH
MAGOSABURO KASAI • College of Agriculture, Kochi University, Kochi, Japan
MARGOT KEARNS • Department of Biochemistry, University of Sydney,
Sydney, Australia
ix
x Contributors
ISMAIL KOLA • Centre for Functional Genomics and Human Disease, Institute
of Reproduction and Development, Monash University, Clayton, Victoria,
Australia
FRANK KONTGEN • The Walter and Eliza Hall Institute of Medical Research,
Melbourne, Australia
MIREILLE H. LAHOUD • Centre for Functional Genomics and Human Disease,
Monash University, Melbourne, Australia
FRANCESCA LAZNER • Centre for Functional Genomics and Human Disease,
Monash University, Melbourne, Australia
CAROL C. LINDBR • The Jackson Laboratory, Bar Harbor, ME
LYNN MAR • Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto, Canada
VIVIENNE S. MARSHALL • Wisconsin Regional Primate Research Center,
University of Wisconsin, Madison, WI
LARRY E. MOBRAATEN • The Jackson Laboratory, Bar Harbor, ME
HUGH MORGAN • Department of Biochemistry, University of Sydney,
Sydney, Australia
ANDRAS NAGY • Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto, Ontario, Canada
ILONA ORMSBY • Department of Molecular Genetics, Biochemistry
and Microbiology, University of Cincinnati College of Medicine,
Cincinnati, OH
UTA PICH • In Vitro Differentiation Group, IPK Gatersleben,
Gatersleben, Germany
GABRIEL A. QUINLAN • Embryology Unit, Children's Medical Research
Institute, Sydney, Australia
PETER D. RATHJEN • Department of Biochemistry, University of Adelaide,
Adelaide, Australia
SUSAN W. REID • Neural Development Group, Mouse Cancer Genetics
Program, NCI-Frederick Cancer Research and Development Center,
Frederick MD
SIKA RISTEVSKI • Centre for Functional Genomics and Human Disease,
Monash University, Melbourne, Australia
L. PHILIP SANFORD • Department of Molecular Genetics, Biochemistry and
Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH
JOHN J. SHARP • The Jackson Laboratory, Bar Harbor, ME
JILLIAN M. SHAW- • Center for Early Human Development, Institute of
Reproduction and Development, Monash University, Melbourne, Australia
DESMOND J. SMITH • Department of Molecular and Medical Pharmacology,
UCLA School of Medicine, Los Angeles, CA