genomic imprinting. methods and protocols

Methods in Molecular Biology TM HUMANA PRESS HUMANA PRESS Methods in Molecular Biology TM Genomic Imprinting Edited by Andrew Ward VOLUME 181 Methods and Protocols Genomic Imprinting Edited by Andrew Ward Methods and Protocols Genomic Imprinting www.dnathink.org guoxzh@dnathink.org 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 195. Quantitative Trait Loci: Methods and Protocols, edited by Nicola J. Camp and Angela Cox, 2002 194. Post-translational Modification Reactions, edited by Christoph Kannicht, 2002 193. RT-PCR Protocols, edited by Joseph 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. 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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 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 v 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 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 vi Preface 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 Contents 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 from Xenopus 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 GAIL ADAM • Eurona Medical AB, Kungsängsvägen, Uppsala, Sweden R OGER L. P. ADAMS • IBLS, University of Glasgow, Scotland, UK S ALLY ADAMS • Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom J USTIN F X. AINSCOUGH • Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, United Kingdom M IRA ARIEL • Department of Cellular Biochemistry and Human Genetics, Hadassah Medical School, The Hebrew University, Jerusalem, Israel M ARISA S. BARTOLOMEI • Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA S HEILA C. BARTON • Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, United Kingdom D ÉBORAH BOURC’HIS • INSERM U383, Hôpital Necker-Enfants Malades, Paris Cedex, France G IOVANNA BRAIDOTTI • Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands M ARIKA CHARALAMBOUS • Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom H ENGMI CUI • Departments of Medicine, Oncology, and Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD W ENDY L. DEAN • Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Programme, The Babraham Institute, Cambridge, United Kingdom G HISLAINE DELL • Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom H UGH G. DICKINSON • Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, United Kingdom O SMAN EL-MAARRI • Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany S ABINE ENGEMANN • Max-Planck-Institut für molekulare Genetik, Ihnestr, Berlin, Germany ix Contributors [...]... been transfected with the neo and leukemia inhibitory factor, LIF, genes, hence is designated SNL For handling ova: Pasteur pipets pulled by hand over a flame to 0.2 and 0.3 mm in outer diameter (od) and flame polished at the tip For disaggregating blastocyst outgrowths: Borosilicate glass capillary tubing, 1 mm od and standard wall thickness, pulled and broken to 0.06 mm od and flame-polished at the tip... in-vivo system of chimera production; and (4) can provide large quantities of cellular material such as DNA for studies of chromatin structure (11,12) Nevertheless, it is From: Methods in Molecular Biology, vol 181: Genomic Imprinting: Methods and Protocols Edited by: A Ward © Humana Press Inc., Totowa, NJ 21 22 Mann important to realize that their derivation and unlimited capacity for division can... involving From: Methods in Molecular Biology, vol 181: Genomic Imprinting: Methods and Protocols Edited by: A Ward © Humana Press Inc., Totowa, NJ 1 2 Dean, Kelsey, and Reik micromanipulation Controlled bispermic fertilization of enucleated metaphase II (MII) oocytes has been used to produce AG embryos to the blastocyst stage (6) However, the more usual route is to enucleate a fertilized embryo and replace... Non-complementation phenomena and their bearing on non-dysfunctional effects In: Aneuploidy: Etiology and mechanisms (Dellarco, K L., Voytele, P E., and Hollaender, A., eds.), Plenum, New York, pp 363–376 15 Beechey, C V and Cattanach, B.M (1998) MRC Genetics Unit, Harwell, Oxfordshire World Wide WebSite Genetic and Physical Imprinting Map of the Mouse (http://www.mgu.har.mrc.ac.uk/imprinting/imprinting.html) 16... embryos by electrofusion Methods Enzymol 225, 919–930 30 Lawitts, J A and Biggers, J D (1993) Culture of preimplantation embryos Methods Enzymol 225, 153–190 31 Hogan, B., Costantini, F., and Lacy, E (1986) Manipulating the Mouse Embryo: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 32 Papaioannou, V and Johnson, R (1993) Production of chimeras and genetically defined... one such factor (2,3) and is indispensable for the propagation of mouse ES cells at least when primary embryo fibroblasts (PEFs) are used as feeder layers (4) A number of features of ES cells make them useful for studying genomic imprinting They (1) are diploid and can be derived such that they contain only maternally and paternally derived genomes, termed parthenogenetic and androgenetic cell lines,... 8, and ref 39) References 1 McGrath, J and Solter, D (1983) Nuclear transplantation in the mouse embryo by microsurgery and cell fusion Science 220, 1300–1303 2 Surani, M A H., Barton, S C., and Norris, M L (1984) Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis Nature 308, 548–550 3 Barton, S C., Surani, M A H., and Norris, M L (1984) Role of paternal and. .. the use of real embryonic material for studying genomic imprinting when possible 2 Materials 1 1× Dulbecco’s phosphate-buffered saline; with and without calcium and magnesium (DPBS+ and DPBS–, respectively) 2 1× trypsin-EDTA solution; 0.25% trypsin (1Ϻ250) and 1 mM ethylenediaminetetraacetic acid (EDTA) in Hank’s balanced salt solution without calcium and magnesium or in DPBS– To 100 mL, add 1 mL of... Y., and Nakahara, T (1993) Development of androgenetic mouse embryos produced by in vitro fertilisation of enucleated oocytes Molec Reprod Dev 34, 43–46 7 McGrath, J and Solter, D (1984) Completion of mouse embryogenesis requires both the maternal and paternal genomes Cell 37, 170–183 Generation of Monoparental Embryos 17 8 O’Neill, Q T., Rolfe, L R., and Kaufman, M H (1991) Developmental potential and. .. 214–219 9 Bos-Mikich, A., Swann, K., and Whittingham, D G (1995) Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes Molec Reprod Dev 41, 84–90 10 Henery, C C and Kaufman, M H (1993) The incidence of aneuploidy after single pulse electroactivation of mouse oocytes Molec Reprod Dev 34, 299–307 11 Moore, T and Haigh, D (1991) Genomic imprinting in mammalian development: . Methods in Molecular Biology TM HUMANA PRESS HUMANA PRESS Methods in Molecular Biology TM Genomic Imprinting Edited by Andrew Ward VOLUME 181 Methods and Protocols Genomic Imprinting Edited. Bacterial Toxins: Methods and Protocols, edited by Otto Holst, 2000 144. Calpain Methods and Protocols, edited by John S. Elce, 2000 143. Protein Structure Prediction: Methods and Protocols, edited. 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