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 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. 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 and Antioxidant Protocols, edited 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 157. Mycotoxin Protocols, edited by Mary W. Trucksess and Albert E. Pohland, 2001 156. Antigen Processing and Presentation Protocols, edited by Joyce C. Solheim, 2001 155. Adipose Tissue Protocols, edited by Gérard Ailhaud, 2000 154. Connexin Methods and Protocols, edited by Roberto Bruzzone and Christian Giaume, 2001 153. Neuropeptide Y Protocols, edited by Ambikaipakan Balasubramaniam, 2000 152. DNA Repair Protocols: Prokaryotic Systems, edited by Patrick Vaughan, 2000 151. Matrix Metalloproteinase Protocols, edited by Ian M. Clark, 2001 150. Complement Methods and Protocols, edited by B. Paul Mor- gan, 2000 149. The ELISA Guidebook, edited by John R. Crowther, 2000 148. DNA–Protein Interactions: Principles and Protocols (2nd ed.), edited by Tom Moss, 2001 147. Affinity Chromatography: Methods and Protocols, edited by Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, and Wolfgang Berthold, 2000 146. Mass Spectrometry of Proteins and Peptides, edited by John R. Chapman, 2000 145. 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 by David Webster, 2000 142. Transforming Growth Factor-Beta Protocols, edited by Philip H. Howe, 2000 141. Plant Hormone Protocols, edited by Gregory A. Tucker and Jeremy A. Roberts, 2000 140. Chaperonin Protocols, edited by Christine Schneider, 2000 139. Extracellular Matrix Protocols, edited by Charles Streuli and Michael Grant, 2000 138. Chemokine Protocols, edited by Amanda E. I. Proudfoot, Timothy N. C. Wells, and Christine Power, 2000 Genomic Imprinting Methods and Protocols Edited by Andrew Ward Department of Biology and Biochemistry University of Bath Claverton Down, Bath, UK 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 © 2002 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. 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. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information published and to describe generally accepted practices. The contributors herein have care- fully checked to ensure that the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time of publication. Notwithstanding, since new research, changes in government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions constantly occur, the reader is advised to check the product information provided by the manufacturer of each drug for any change in dosages or for additional warnings and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug. It is the responsibility of the treating physician to determine dosages and treatment strategies for individual patients. Further, it is the responsi- bility of the health care provider to ascertain the Food and Drug Administration status of each drug or device used in their clinical practice. The publishers, editors, and authors are not responsible for errors or omissions or for any consequences from the application of the information presented in this book and make no warranty, express or implied, with respect to the contents in this publication. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by Patricia F. Cleary. Cover illustration: In situ hybridization showing the expression pattern of the Igf2 gene in a mouse embryo. Artwork courtesy of Dr. Andrew Ward and Dr. Marika Charalambous. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: humana@humanapr.com or visit our website at http://humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-741-X/02 $10.00 + $00.25]. 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 [...]... (Miles, Inc., Diagnostics Division, cat no 8 2-0 4 7-3 , Kankakee, IL, USA) in cell culturegrade water and filter-sterilize Store 1-mL aliquots at –20°C 3 1000× 2-mercaptoethanol solution; add 70 µL of 2-mercaptoethanol to 10 mL of DPBS– to give 0.1 M solution Mix, then filter-sterilize Store at 4°C and discard after 3 wk 4 1× gelatin solution; add 0.5 g cell culture-grade gelatin to 500 mL DPBS– in a glass... of 4N-PG-4N Aggregation Chimeras 2.4.3.1 PREPARATION OF DISHES FOR AGGREGATION 1 2 3 4 Darning needle KSOM (see Subheading 2.2.2.), equilibrated at 37°C in 5% CO2 under oil Mineral oil Sterile plastic Petri dishes (50 mm) 2.4.3.2 REMOVAL OF ZONA PELLUCIDA 1 Acid Tyrode’s (Sigma: embryo-tested T-1788) 2 FHM (see Subheading 2.2.2.) 3 Drawn 9-in Pasteur pipets (0.2 mm; flame polished) 2.4.3.3 4N-PG-4N “SANDWICH”... Superovulation 1 2 3 4 3- to 4-wk-old female B6CB-F1 (Harlan; Charles River, U.K.) (see Note 1) Phosphate-buffered saline (PBS; Sigma) Pregnant mare serum gonadotropin (PMS-Folligon), 1500 IU per vial (Intervet) Human chorionic gonadotropin (hCG—Chorulon) 500 IU per vial (Intervet) 2.2.2 Collection and Culture of Fertilized Zygotes 1 Dissecting tools; No 5 watch maker’s forceps; fine-pointed scissors (iris;... karyoplast fusion using HVJ or Sendai virus-assisted fusion (1) These experiments laid the foundation for the discovery and exploration of this unique form of non-Mendelian mammalian gene regulation whereby expression of genes and hence phenotype were dictated by the parent from whom they where inherited This parent-of-origin phenomenon is known as genomic imprinting 1.1 Androgenetic Embryos Uniparental... 1181 1-0 31) in 40 mL of DPBS– to achieve a concentration of 17.5 mg/mL active weight; proportion of active weight or microbiological potency is stated on bottle Store as 1.5-mL aliquots in 2-mL sterile screw-capped tubes (Sarstedt, cat no 72.693.005) at –20°C Thawed tubes are stable at 4°C for at least 1 mo 100× mitomycin C solution: dissolve 2 mg in 4.0 mL of DPBS– Store 0.12-mL aliquots in sterile 2-mL... 4°C for months Add 0.2 mL to approx 1.8 mL medium M2 in a 3-cm Petri dish to give 1x solution Mouth-controlled aspiration device to control flow of medium in pipets described above (17) Thin-diameter tubing can be attached to accommodate 1-mm-od glass capillaries Ovum culture dish: Drops of medium CZB (18) of approx 10 µL pipetted in rows onto a 3-cm Petri dish, then overlaid with mineral oil These dishes... aggregation chimeras are produced with PG embryos at the 8-cell and the tetraploid components at the “4-cell” stage The tetraploid embryos will “compact” after the “4-cell” stage, as this is equivalent to the diploid 8-cell stage when compaction occurs normally Thus, the aggregates must be made early on the day after fusion 1 Prepare aggregation depression in 50-mm bacteriological dishes using a darning needle... (1991) Genomic imprinting in mammalian development: a parental tug-of-war Trends Genet 7, 45–49 12 Sturm, K S., Flannery, M L., and Pedersen, R A (1994) Abnormal development of embryonic and extraembryonic cell lineages in parthenogenetic mouse embryos Dev Dynam 210, 11–28 13 Spindle, A., Sturm, K S., Flannery, M., Meneses, S J., Wu, K., and Pedersen, R A (1996) Defective chorioallantoic fusion in mid-gestation... (1985) 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)... glucose in embryo culture-grade H2O (BDH) 30-mm sterile Petri dishes 5 µg/mL CCB (see Subheading 2.3.2.1.) in KSOM (see Subheading 2.2.2.), equilibrated at 37°C in 5% CO2 under oil 6 KSOM (see Subheading 2.2.2.), equilibrated at 37°C in 5% CO2 under oil 2.4.2 Production of Tetraploid Embryos 2.4.2.1 RECOVERY OF TWO-CELL EMBRYOS 1 2 3 4 5 6 Nonbeveled 33-gage flushing needles Sterile 2-mL disposable syringes . 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 M E T H O D. by Andrew Ward. p. cm. (Methods in molecular biology ; v. 181) Includes bibliographical references and index. ISBN 0-8 960 3-7 41-X (alk. paper) 1. Genomic imprinting Laboratory manuals. I. Ward, . Transactional Reporting Service is: [ 0-8 960 3-7 41-X/02 $10.00 + $00.25]. 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