Genomic Imprinting: Methods and Protocols

389 Pages·2002·1.951 MB·English

by  Wendy L. Dean, Gavin Kelsey, Wolf Reik (auth.), Andrew Ward (eds.)

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M E T H O D S I N M O L E C U L A R B I O L O G YTM
Genomic
Imprinting
Methods and Protocols
Edited by
Andrew Ward
Department of Biology and Biochemistry
University of Bath
Claverton Down, Bath, UK
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Genomic imprinting: methods and protocols / edited by Andrew Ward.
p. cm. -- (Methods in molecular biology ; v. 181)
Includes bibliographical references and index.
ISBN 0-89603-741-X (alk. paper)
1. Genomic imprinting--Laboratory manuals. I. Ward, Andrew, 1964- II. Series.
QH 450 .G468 2001
572.8'6'028--dc21
2001024456
Preface
Genomic imprinting is the process by which gene activity is regulated
according to parent of origin. Usually, this means that either the maternally
inherited or the paternally inherited allele of a gene is expressed while the
opposite allele is repressed. The phenomenon is largely restricted to mammals
and flowering plants and was first recognized at the level of whole genomes.
Nuclear transplantation experiments carried out in mice in the late 1970s
established the non-equivalence of the maternal and paternal genomes in
mammals, and a similar conclusion was drawn from studies of interploidy
crosses of flowering plants that extend back to at least the 1930s. Further mouse
genetic studies, involving animals carrying balanced translocations (reviewed
in Chapter 3), indicated that imprinted genes were likely to be widely scattered
and would form a minority within the mammalian genome. The first imprinted
genes were identified in the early 1990s; over forty are now known in mammals
and the list continues steadily to expand.
Genomic Imprinting: Methods and Protocols aims to collect protocols
that have been applied to the study of imprinting or imprinted genes. Many of
the protocols are based on more widely used embryology or molecular biology
techniques that have been adapted for imprinting research. All of the included
methods remain gainfully employed in either (or both) the discovery or analysis
of imprinted genes. Chapter 1 describes the nuclear transplantation methods,
first used in the 1970s, for the generation of mouse embryos with genomes of
entirely maternal or entirely paternal origin. The first five chapters are specific to
the mouse, though some of the principles could be applied to other species.
For instance, the techniques described in Chapters 4 and 5 for generating
transgenic mice using large fragments of genomic DNA have resulted in several
examples of the faithful reproduction of imprinted gene expression at ectopic
loci. The first few imprinted genes have recently been identified in plants and
it will be interesting to know whether the imprinting of these genes can be
similarly reproduced within plant transgenes.
The majority of protocols describe molecular techniques and most of
these allow examination of gene structure or expression in an allele-specific
manner, which is an essential aspect of most imprinting studies. Protocols are
v
vi Preface
included for identifying imprinted genes (Chapters 6–8), for analyzing
imprinted gene expression (Chapters 9–12), for the study of DNA methylation
and methylation-sensitive DNA-binding proteins (Chapters 13–20), and for
examining chromatin structure (Chapters 21–24). The final chapter is a review of
genomic imprinting in plants. Although imprinting must have arisen indepen-
dently in plants and animals, the available evidence suggests that the imprinting
mechanisms in these species may share common features, such as the involve-
ment of DNA methylation in distinguishing maternal and paternal alleles. Thus,
the molecular methods that are already extensively used to study mammalian
imprinted genes will surely find even wider employment as the genomic
imprinting field continues to expand.
I thank all of the authors for their outstanding contributions to this volume.
On behalf of us all I extend the hope that this effort to make these methods
accessible will prove useful to genomic imprinting aficionados everywhere.
Andrew Ward
Contents
Preface ............................................................................................................v
Contributors....................................................................................................ix
1 Generation of Monoparental Embryos
for Investigation into Genomic Imprinting
Wendy L. Dean, Gavin Kelsey, and Wolf Reik...................................1
2 Deriving and Propagating Mouse Embryonic Stem Cell Lines
for Studying Genomic Imprinting
Jeffrey R. Mann....................................................................................21
3 Balanced Translocations for the Analysis
of Imprinted Regions of the Mouse Genome
Anne C. Ferguson-Smith, Maxine Tevendale,
Pantelis Georgiades, and Valerie Grandjean..............................41
4 Production of YAC Transgenic Mice by Pronuclear Injection
Justin F.-X. Ainscough, Rosalind M. John,
and Sheila C. Barton.......................................................................55
5 A Transgenic Approach to Studying Imprinted Genes:
Modified BACs and PACs
Rosalind M. John, Justin F.-X. Ainscough, and Sheila C. Barton....67
6 Methylation-Sensitive Genome Scanning
Izuho Hatada and Tsunehiro Mukai...................................................83
7 Subtraction-Hybridization Method for the Identification
of Imprinted Genes
Fumitoshi Ishino, Yoshimi Kuroiwa, Naoki Miyoshi, Shin
Kobayashi, Takashi Kohda, and Tomoko Kaneko-Ishino........101
8 Identification of Imprinted Loci by Methylation: Use of Methylation-
Sensitive Representational Difference Analysis (Me-RDA)
Rachel J. Smith and Gavin Kelsey..................................................113
9 Ribonuclease Protection
Joanne L. Thorvaldsen and Marisa S. Bartolomei........................133
10 Quantitative RT-PCR-Based Analysis of Allele-Specific
Gene Expression
Judith Singer-Sam and Chunguang Gao........................................145
11 Allele-Specific In Situ Hybridization (ASISH)
Rolf Ohlsson, Kristian Svensson, Hengmi Cui,
Helena Malmikumpu, and Gail Adam..........................................153
vii
viii Contents
12 RNA-FISH to Analyze Allele-Specific Expression
Giovanna Braidotti.............................................................................169
13 Flow Cytometry and FISH to Investigate Allele-Specific Replication
Timing and Homologous Association of Imprinted Chromosomes
Janine LaSalle and Marc Lalande....................................................181
14 Southern Analysis Using Methyl-Sensitive Restriction Enzymes
Tom Moore..........................................................................................193
15 A PCR-Based Method for Studying DNA Methylation
Mira Ariel.............................................................................................205
16 Bisulfite-Based Methylation Analysis of Imprinted Genes
Sabine Engemann, Osman El-Maarri, Petra Hajkova,
Joachim Oswald, and Joern Walter............................................217
17 Direct Analysis of Chromosome Methylation
Déborah Bourc’his and Evani Viegas-Péquignot..........................229
18 In Vitro Methylation of Predetermined Regions
in Recombinant DNA Constructs
Ilse Van den Wyngaert, Roger L. P. Adams,
and Stefan U. Kass........................................................................243
19 In Vitro Methylation of Specific Regions in Recombinant DNA
Constructs by Excision and Religation
Ghislaine Dell, Marika Charalambous, and Andrew Ward...........251
20 Detection of Methyl-Sensitive DNA-Binding Proteins
with Possible Involvement in the Imprinting Phenomenon
Kerstin Otteand Björn Rozell...........................................................259
21 Probing Chromatin Structure with Nuclease Sensitivity Assays
Richard I. Gregory, Sanjeev Khosla, and Robert Feil...................269
22 Examining Histone Acetylation at Specific Genomic Regions
Ji-Fan Hu and Andrew R. Hoffman..................................................285
23 Purification of the MeCP2/Histone Deacetylase Complex
fromXenopus laevis
Peter L. Jones, Paul A. Wade, and Alan P. Wolffe.......................297
24 Reconstitution of Chromatin In Vitro
Kiyoe Uraand Yasufumi Kaneda.....................................................309
25 Genomic Imprinting in Plants
Rinke Vinkenoog, Melissa Spielman, Sally Adams,
Hugh G. Dickinson, and Rod J. Scott.........................................327
Index............................................................................................................371
Contributors
GAIL ADAM• Eurona Medical AB, Kungsängsvägen, Uppsala, Sweden
ROGER L. P. ADAMS • IBLS, University of Glasgow, Scotland, UK
SALLY ADAMS • Department of Biology and Biochemistry, University
of Bath, Claverton Down, Bath, United Kingdom
JUSTIN F.-X. AINSCOUGH • Wellcome/CRC Institute of Cancer
and Developmental Biology, University of Cambridge, Cambridge,
United Kingdom
MIRA ARIEL • Department of Cellular Biochemistry and Human Genetics,
Hadassah Medical School, The Hebrew University, Jerusalem, Israel
MARISA S. BARTOLOMEI • Howard Hughes Medical Institute
and Department of Cell and Developmental Biology, University
of Pennsylvania School of Medicine, Philadelphia, PA
SHEILA C. BARTON• Wellcome/CRC Institute of Cancer and Developmental
Biology, University of Cambridge, Cambridge, United Kingdom
DÉBORAH BOURC’HIS• INSERM U383, Hôpital Necker-Enfants Malades,
Paris Cedex, France
GIOVANNA BRAIDOTTI • Department of Cell Biology and Genetics,
Erasmus University, Rotterdam, The Netherlands
MARIKA CHARALAMBOUS • Department of Biology and Biochemistry,
University of Bath, Claverton Down, Bath, United Kingdom
HENGMI CUI • Departments of Medicine, Oncology, and Molecular
Biology and Genetics, Johns Hopkins University School of Medicine,
Baltimore, MD
WENDY L. DEAN• Laboratory of Developmental Genetics and Imprinting,
Developmental Genetics Programme, The Babraham Institute,
Cambridge, United Kingdom
GHISLAINE DELL • Department of Biology and Biochemistry, University
of Bath, Claverton Down, Bath, United Kingdom
HUGH G. DICKINSON • Department of Plant Sciences, University of Oxford,
South Parks Road, Oxford, United Kingdom
OSMAN EL-MAARRI • Institute of Experimental Haematology
and Transfusion Medicine, Bonn, Germany
SABINE ENGEMANN • Max-Planck-Institut für molekulare Genetik, Ihnestr,
Berlin, Germany
ix