Methods in Molecular Biology TM TM VOLUME 185 Embryonic Stem Cells Methods and Protocols Edited by Kursad Turksen HUMANA PRESS Embryonic Stem Cells 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, SERIES EDITOR 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 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 Morgan, 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 137 Developmental Biology Protocols, Volume III, edited by Rocky S Tuan and Cecilia W Lo, 2000 136 Developmental Biology Protocols, Volume II, edited by Rocky S Tuan and Cecilia W Lo, 2000 135 Developmental Biology Protocols, Volume I, edited by Rocky S Tuan and Cecilia W Lo, 2000 134 T Cell Protocols: Development and Activation, edited by Kelly P Kearse, 2000 133 Gene Targeting Protocols, edited by Eric B Kmiec, 2000 132 Bioinformatics Methods and Protocols, edited by Stephen Misener and Stephen A Krawetz, 2000 131 Flavoprotein Protocols, edited by S K Chapman and G A Reid, 1999 130 Transcription Factor Protocols, edited by Martin J Tymms, 2000 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 D D D TM Embryonic Stem Cells Methods and Protocols Edited by Kursad Turksen Ottawa Health Research Institute, Ottawa, Ontario, Canada Humana Press Totowa, New Jersey © 2002 Humana Press Inc 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 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 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 This publication is printed on acid-free paper ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials Production Editor: Diana Mezzina Cover design by Patricia F Cleary 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: www.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: [0-89603-8815/02 (hardcover) $10.00 + $00.25] Printed in the United States of America 10 Library of Congress Cataloging in Publication Data Embryonic Stem Cells: methods and protocols / edited by Kursad Turksen p cm (Methods in molecular biology ; v 185) Includes bibliographical references and index ISBN 0-89603-881-5 (alk paper) Embryonic Stem Cells Laboratory manuals I Turksen, Kursad II Series QH440.5 E43 2002 612'.0181 dc21 2001026459 Preface It is fair to say that embryonic stem (ES) cells have taken their place beside the human genome project as one of the most discussed biomedical issues of the day It also seems certain that as this millennium unfolds we will see an increase in scientific and ethical debate about their potential utility in society On the scientific front, it is clear that work on ES cells has already generated new possibilities and stimulated development of new strategies for increasing our understanding of cell lineages and differentiation It is not naïve to think that, within a decade or so, our overall understanding of stem cell biology will be as revolutionized as it was when the pioneering hemopoietic stem cell studies of Till and McCulloch in Toronto captured our imaginations in 1961 With it will come better methods for ES and lineage-specific stem cell identification, maintenance, and controlled fate selection Clearly, ES cell models are already providing opportunities for the establishment of limitless sources of specific cell populations In recognition of the growing excitement and potential of ES cells as models for both the advancement of basic science and future clinical applications, I felt it timely to edit this collection of protocols (Embryonic Stem Cells) in which forefront investigators would provide detailed methods for use of ES cells to study various lineages and tissue types We are pleased to provide Embryonic Stem Cells: Methods and Protocols, a broadscaled work of 35 chapters containing step-by-step protocols suitable for use by both experienced investigators and novices in various ES cell technologies In the first section of the volume, there are chapters with detailed protocols for ES cell isolation, maintenance, modulation of gene expression, and studies of ES cell cycle and apoptosis Embryonic Stem Cells also includes chapters with protocols for the use of ES cells to generate diverse cell and tissue types, including blood, endothelium, adipocytes, skeletal muscle, cardiac muscle, neurons, osteoclasts, melanocytes, keratinocytes, and hair follicle cells The second part of the volume contains a series of cutting edge techniques that have already been shown to have, or will soon have, tremendous utility with ES cells and their differentiated progeny These chapters include the use of cDNA arrays in gene expression analysis, phage display antibody libraries to generate antibodies against very rare antigens, and phage display libraries to identify and characterize protein and protein interactions, to name a few Collectively, these protocols should prove a useful resource not only to those who are using or wish to use ES cells to study fate choices and specific lineages, but also to those interested in cell and developmental biology more generally We hope that this book will also serve as a catalyst spurring others to use ES cells for lineages not yet being widely studied with this model and to develop new methodologies that would contribute to both the fundamental understanding of stem cells and their potential utility v vi Preface Embryonic Stem Cells would not have materialized at all had the contributors not recognized the special value of disseminating their protocols and hard-won expertise I am extremely grateful to them for their commitment, dedication, and promptness with submissions! I am also grateful to Dr John Walker for having faith in and supporting me throughout this project I wish also to acknowledge the great support provided by many at Humana Press, specifically Elyse O'Grady, Craig Adams, Diana Mezzina, and Tom Lanigan A special thank you goes to my dedicated coworker, Tammy-Claire Troy, who, with her infectious optimism and tireless commitment, became a crucial factor in the editing and completion of the volume I am grateful to N Urfe, P Kael, and M Chambers for their unintentional “awesome” contributions Finally, I hope that the volume will achieve the intent that I had originally imagined: that it will prove a volume with something for both experts and novices alike, that it will serve as a launching point for further developments in stem cells, and that we will all-too-soon wish to expand and update it with other emerging concepts, insights and methods! Kursad Turksen Contents Preface v Contributors xi Color Plates xv Methods for the Isolation and Maintenance of Murine Embryonic Stem Cells Marsha L Roach and John D McNeish The Use of Chemically Defined Media for the Analyses of Early Development in ES Cells and Mouse Embryos 10 Gabriele Proetzel and Michael V Wiles 17 Analysis of the Cell Cycle in Mouse Embryonic Stem Cells Pierre Savatier, Hélène Lapillonne, Ludmila Jirmanova, Luigi Vitelli, and Jacques Samarut 27 Murine Embryonic Stem Cells as a Model for Stress Proteins and Apoptosis During Differentiation André-Patrick Arrigo and Patrick Mehlen 35 Effects of Altered Gene Expression on ES Cell Differentiation Yong Fan and J Richard Chaillet 45 Hypoxic Gene Regulation in Differentiating ES Cells David M Adelman and M Celeste Simon 55 Regulation of Gap Junction Protein (Connexin) Genes and Function in Differentiating ES Cells Masahito Oyamada, Yumiko Oyamada, Tomoyuki Kaneko, and Tetsuro Takamatsu 63 Embryonic Stem Cell Differentiation as a Model to Study Hematopoietic and Endothelial Cell Development Stuart T Fraser, Minetaro Ogawa, Satomi Nishikawa, and Shin-Ichi Nishikawa 71 Analysis of Bcr-Abl Function Using an In Vitro Embryonic Stem Cell Differentiation System Takumi Era, Stephane Wong, and Owen N Witte 83 Embryonic Stem Cells as a Model for Studying Osteoclast Lineage Development Toshiyuki Yamane, Takahiro Kunisada, and Shin-Ichi Hayashi 97 vii viii Contents 11 Differentiation of Embryonic Stem Cells as a Model to Study Gene Function During the Development of Adipose Cells Christian Dani 107 12 Embryonic Stem Cell Differentiation and the Vascular Lineage Victoria L Bautch 117 13 Embryonic Stem Cells as a Model to Study Cardiac, Skeletal Muscle, and Vascular Smooth Muscle Cell Differentiation Anna M Wobus, Kaomei Guan, Huang-Tian Yang, and Kenneth R Boheler 127 14 Cardiomyocyte Enrichment in Differentiating ES Cell Cultures: Strategies and Applications Kishore B S Pasumarthi and Loren J Field 157 15 Embryonic Stem Cells as a Model for the Physiological Analysis of the Cardiovascular System Jürgen Hescheler, Maria Wartenberg, Bernd K Fleischmann, Kathrin Banach, Helmut Acker, and Heinrich Sauer 169 16 Isolation of Lineage-Restricted Neural Precursors from Cultured ES Cells Tahmina Mujtaba and Mahendra S Rao 189 17 Lineage Selection for Generation and Amplification of Neural Precursor Cells Meng Li 205 18 Selective Neural Induction from ES Cells by Stromal Cell-Derived Inducing Activity and Its Potential Therapeutic Application in Parkinson's Disease Hiroshi Kawasaki, Kenji Mizuseki, and Yoshiki Sasai 217 19 Epidermal Lineage Tammy-Claire Troy and Kursad Turksen 229 20 ES Cell Differentiation Into the Hair Follicle Lineage In Vitro Tammy-Claire Troy and Kursad Turksen 255 21 Embryonic Stem Cells as a Model for Studying Melanocyte Development Toshiyuki Yamane, Shin-Ichi Hayashi, and Takahiro Kunisada 261 22 Using Progenitor Cells and Gene Chips to Define Genetic Pathways S Steven Potter, M Todd Valerius, and Eric W Brunskill 269 23 ES Cell-Mediated Conditional Transgenesis Marina Gertsenstein, Corrinne Lobe and Andras Nagy 285 24 Switching on Lineage Tracers Using Site-Specific Recombination Susan M Dymecki, Carolyn I Rodriguez, and Rajeshwar B Awatramani 309 Contents ix 25 From ES Cells to Mice: The Gene Trap Approach Francesco Cecconi and Peter Gruss 335 26 Functional Genomics by Gene-Trapping in Embryonic Stem Cells Thomas Floss and Wolfgang Wurst 347 27 Phage-Displayed Antibodies to Detect Cell Markers Jun Lu and Steven R Sloan 381 28 Gene Transfer Using Targeted Filamentous Bacteriophage David Larocca, Kristen Jensen-Pergakes, Michael A Burg, and Andrew Baird 393 29 Single-Cell PCR Methods for Studying Stem Cells and Progenitors Jane E Aubin, Fina Liu, and G Antonio Candeliere 403 30 Nonradioactive Labeling and Detection of mRNAs Hybridized onto Nucleic Acid cDNA Arrays Thorsten Hoevel and Manfred Kubbies 417 31 Expression Profiling Using Quantitative Hybridization on Macroarrays Geneviève Piétu and Charles Decraene 425 32 Isolation of Antigen-Specific Intracellular Antibody Fragments as Single Chain Fv for Use in Mammalian Cells Eric Tse, Grace Chung, and Terence H Rabbitts 433 33 Detection and Visualization of Protein Interactions with Protein Fragment Complementation Assays Ingrid Remy, André Galarneau, and Stephen W Michnick 447 34 Direct Selection of cDNAs by Phage Display Reto Crameri, Gernot Achatz, Michael Weichel, and Claudio Rhyner 461 35 Screening for Protein–Protein Interactions in the Yeast Two-Hybrid System in Embryonic Stem Cells R Daniel Gietz and Robin A Woods 471 Index 487 486 Gietz and Woods 21 Tanahashi, H and Tabira, T (2000) Alkaline treatment after X-gal staining reaction for Escherichia coli β-galactosidase enhances sensitivity Anal Biochem 279, 122–123 22 Hoffman, C S and Winston, F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli Gene 57, 267–272 23 El Hounsni, H., Vandenbroere, I., Perez-Morga, D., Christophe, D., and Pirson, I (1998) A rare case of false positive in a yeast two-hybrid screening: the selection of rearranged bait constructs that produce a functional Gal4 activity Anal Biochem 262, 94–96 24 Miller, J H (1972) Experiments in Molecular Genetics CHS Laboratory Press, Cold Spring Harbor, N.Y 25 Bartel, P L., Chien, C.-T., Sternglanz, R., and Fields, S (1993) Elimination of false positives that arise in using the two-hybrid system Biotechniques 14, 920–924 26 Cormack, R S and Somssich, I E (1997) Dampening of bait proteins in the two-hybrid system Anal Biochem 248, 184–186 Index 487 Index A Activin, embryonic stem cell differentiation marker, 22 Adipocyte, induction from embryonic stem cells, cell maintenance, 109–111, 113, 114 embryoid body differentiation, 111, 112, 115 materials, 109, 110, 113, 114 Oil Red O staining, 109, 110, 112, 113 requirements for lineage commitment, 107, 108 reverse transcription-polymerase chain reaction analysis of gene expression during differentiation, annealing temperature, 113, 114 primers, 110 RNA preparation, 110, 113, 115 X-Gal staining, 109, 110, 112, 113 Antibody, phage display (see Phage display) single chain Fv, applications, 433, 434 intracellular antibody capture technology, 434 isolation of intracellular fragments binding to specific antigens, β−galactosidase filter assay, 437, 442 materials, 435–438 overview, 435 phage library screening with antigen, 435, 436, 438, 439, 445 plasmid extraction from yeast, 438, 442, 443 polymerase chain reaction, 438, 443, 444 sublibrary preparation, 436, 439, 440, 445 487 transformation of bacteria, 436, 441 Western blot, 438, 444 yeast antigen–antibody interaction screening, 436–438, 441–443, 445 yeast expression library, 436, 440, 445 Apoptosis, detection during embryonic stem cell differentiation, 42, 44 DNA fragmentation analysis, 42 B Bcr-Abl fusion, chronic myelogenous leukemia role, 83 immortalization of cells, 83 tetracycline-regulated gene expression analysis of embryonic stem cell hematopoiesis, Bcr-Abl clone establishment, 89, 90, 93 differentiation induction, 86, 90–93 embryonic stem cell maintenance, 85–88, 93 materials, 85, 86, 93 OP9 stromal cell line maintenance, 86, 90, 93 overview, 83–85 Tet-off parental cell line generation, 88, 89 vectors, 86, 87 BMPs (see Bone morphogenetic proteins) Bone morphogenetic proteins (BMPs), embryonic stem cell differentiation marker, 23 neural induction in ectoderm, 217, 218 BrdU (see Bromodeoxyuridine) Bromodeoxyuridine (BrdU), cell cycle analysis, 31 488 C Cardiomyocyte, embryonic stem cell differentiation, advantages of differentiated culture, 157, 158 culture, cell lines, 128 feeder layers, 133 media, 128, 129, 149 undifferentiated embryonic stem cells, 134, 149 differentiation culture, 134, 136, 149 embryoid body cardiomyogenesis (see Embryoid body) equipment, 132, 133 factors affecting, 128 genetic enrichment approach, advantages, 159, 167 colony growth assay, 164–167 differentiation conditions, 161, 162, 167 graft generation, 162–164 materials, 159, 161 overview, 158, 159 periodic acid Schiff's staining, 159, 162, 167 selection of differentiated cells, 162, 167 transfection and selection, 161 immunofluorescence analysis, 130, 139, 141, 150 overview, 127 pharmacological analysis, 141 primary culture limitations, 157 reverse transcription-polymerase chain reaction, amplification reactions, 138, 150 cell sample preparation, 137, 149 gel electrophoresis of products, 139 materials, 129, 130, 149 primers and genes, 140 reverse transcription, 138, 150 RNA isolation, 137, 138, 150 single cell isolation, 131, 139 transfection, analysis, DNA isolation, 131, 145, 146 polymerase chain reaction, 131, 132, 146, 151 Southern blot, 132, 148, 151 clone selection, 145, 150 DNA preparation, stable transfections, 143, 150, 151 Index transient transfection, 143, 150, 151 electroporation, 143–145, 150 gain-of-function and loss-of-function analysis, 148, 149 materials, 131 rationale for differentiation studies, 141, 143 Cell cycle, embryonic stem cells, bromodeoxyuridine analysis, 31 flow cytometry, 30–32 Hsp27 effects and assay, 41, 42 p53 pathway, 28, 29 phases, 27 retinoblastoma pathway, 27, 28 synchronization by mitotic shake-off, 29–32 Chronic myelogenous leukemia (CML) (see Bcr-Abl fusion) CML (see Chronic myelogenous leukemia) Complementary DNA array hybridization, digoxygenin detection, 422 fibroblast cell culture, 420, 422 hybridization conditions, 421, 423 macroarrays (see Macroarray hybridization) materials, 419 membrane array reuse, 422 messenger RNA, enrichment, 420, 421 extraction, 420, 422, 423 labeling with DIGChemLink, 421, 423 overview, 417–419 RNA extraction, 420, 422, 423 Confocal laser scanning microscopy, optical probing of embryoid bodies, 174, 175 Connexin, embryonic stem cell expression in differentiation, immunofluorescent labeling, 64, 65, 67–69 reverse transcription-polymerase chain reaction, amplification reaction, 66 cDNA synthesis, 66, 69 materials, 63, 64 RNA extraction, 65, 66 gap junction, calcium flux imaging, 65, 68, 69 function, 63 Index Luifer yellow dye coupling assay, 65 Culture, embryonic stem cells of mouse, feeder layer preparation, 3, feeding, 9, 10 freezing, 10 gelatin-coated dish preparation, isolation from inner cell mass, blastocyst culture, blastocyst stage embryo preparation, culture, 4–7 materials, 2, media preparation, overview of conditions, 1, 14, 15 subculture, 9, 10 thawing, D DHFR (see Dihydrofolate reductase) Differentiation, embryonic stem cells, adipocytes (see Adipocyte) chemically defined media for analysis, advantages and limitations, 17–19 cell preparation, 20, 24 egg cylinder embryo culture, 21, 25 hanging drop culture differentiation, 20, 24, 25 hanging drop culture followed by attachment culture differentiation, 21, 25 media preparation, 19, 20 reverse transcription polymerase chain reaction for assessment, activin, 22 bone morphogenetic proteins, 23 Pax6, 22, 23 Rex-1, 22 suspension culture differentiation, 20, 24 whole-mount in situ hybridization, 24 endothelial cells, induction, assay, 75, 78 culture, 75, 77 materials, 74, 75, 77 principles, 72, 73 overview, 71, 72 epidermis (see Epidermal stem cell) gene expression studies (see Connexin; Hsp27; Pem) hair follicle lineage (see Hair follicle cell) hematopoietic cells, induction, culture, 76–79 489 materials, 74, 75, 77 principles, 73, 74 overview, 72 history of study, 17, 18 in vitro differentiation, human cells, 19 mouse cell characterization, melanocytes (see Melanocyte) mesodermal cells, induction, culture, 75, 77 materials, 74, 77 principles, 72 overview, 71, 72 muscle (see Cardiomyocyte; Skeletal muscle cell; Vascular smooth muscle cell) neural induction (see Glial-restricted precursor; Neuronal-restricted precursor; stromal cell-derived inducing activity) osteoclasts (see Osteoclast) tetracycline-regulated gene expression analysis of hematopoiesis, Bcr-Abl clone establishment, 89, 90, 93 differentiation induction, 86, 90–93 embryonic stem cell maintenance, 85–88, 93 materials, 85, 86, 93 OP9 stromal cell line maintenance, 86, 90, 93 overview, 83–85 Tet-off parental cell line generation, 88, 89 vectors, 86, 87 vasculature development, adhesion molecule immunolocalization, fixation, 120, 122, 124 imaging, 120, 121, 123, 124 staining, 120, 122 cystoid embryoid body formation, 117 differentiation, 120–123 embryonic stem cell culture, 120, 121, 123 enzymatic disruption, 120–123 markers, 117, 118 materials, 119–121 mutation studies in validation, 118, 119 490 Dihydrofolate reductase (DHFR) (see Protein-fragment complementation assay) E EB (see Embryoid body) EG cell (see Embryonic germ cell) Electroporation, mouse embryonic stem cells, cell preparation, 10, 11 colony picking, 12, 15 electroporation conditions, 11, 12, 15 expansion into clonal cell lines, 12, 13 Embryoid body (EB), cardiomyogenesis, applications, 170 cardiomyocyte analysis, calcium imaging, 180, 181 intracellular dialysis studies, 181 isolation of cells, 179, 180, 184, 185 materials, 171, 172 patch-clamp, 178–180, 185 embryonic stem cell maintenance, 170, 171 fluorescence probing, cell dissociation, 175, 176 confocal laser scanning microscopy, 174, 175 correction for light attenuation, 176 dynamics analysis, 177, 178 fluorescence distribution coefficient determination, 176 materials, 170–172 micro-electrode array studies, electrophysiological recording, 182 excitability development in cell clusters, 182–184 overview, 181, 182 tissue culture, 182 overview, 169, 170 spinner flask culture, cleaning of spinner flask, 172, 173 efficiency, 172 inoculation, 173 spinner flasks and preparation, 171, 172 epithelial differentiation (see Epidermal stem cell) hypoxia studies, colony-forming unit scoring, 60, 61 Index culture from stem cells, 58–61 disaggregation and replating of progenitors, 59–61 methylcellulose for differentiation and replating, 57 oxygen gradients, 55, 56 Pem expression effects, analysis, cell mixing experiments, 50, 51 growth, 49, 50 morphology, 50 tissue-specific gene expression, 51 structure, 230 Embryonic germ (EG) cell, embryonic stem cell comparison, 189 Embryonic stem (ES) cell, cell cycle analysis (see Cell cycle) embryonic stem cells characterization of mouse cells, gene targeting ability and germline transmission, in vitro differentiation, karyotyping, 1, mycoplasma and viral contamination testing, sex determination, 7, connexin in differentiation (see Connexin) culture (see Culture), embryonic stem cells developmental gene screening (see Complementary DNA array hybridization; Gene chip array; Gene trap; Macroarray hybridization) differentiation (see Differentiation) embryonic stem cells electroporation (see Electroporation) gene trapping (see Gene trap) history of study, 1, 17 Hsp27 expression (see Hsp27) hypoxia effects (see Hypoxia) Pem expression (see Pem) single-cell polymerase chain reaction (see Polymerase chain reaction) transgenic mouse construction (see Transgenic mouse) whole plate shake-off for aggregation or microinjection into embryos, 13–15 Index Endothelial cell, embryonic stem cell differentiation, 218 Endothelial cell, embryonic stem cell differentiation, induction, assay, 75, 78 culture, 75, 77 materials, 74, 75, 77 principles, 72, 73 overview, 71, 72 Epidermal stem cell (see also Hair follicle) cell embryonic stem cell differentiation, alkaline phosphatase histochemistry, 238 Ayoub Shklar staining for keratin, 233, 248 immunofluorescence, 233, 234, 252 induction of differentiation, culture stages, 238, 240 differentiating embryoid body formation, 242 embryoid body formation, 240, 241 epithelial progenitor cell culture, 242–245 mouse keratinocyte feeder layers, 245 in situ hybridization of embryoid bodies, gene selection for screening, 245, 246 materials, 232, 233 riboprobe preparation, 246 whole-mount hybridization, 246, 247, 252 materials, 230–234, 251, 252 reverse transcription-polymerase chain reaction, amplification reactions, 250 DNA removal, 249 materials, 234 primers, 250, 251 reverse transcription, 250 RNA extraction, 248, 249 tissue culture, embryonic fibroblast feeder layers, 235, 236 embryonic stem cell maintenance, 238 491 equipment, 231, 232 freezing cells, 234, 235, 252 materials, 230, 231 media, 231 mitomycin C treatment of feeder layers, 236, 238 thawing cells, 235, 252 epidermis layers, 229, 238 keratin markers of differentiation, 229 stromal cell-derived inducing activity in differentiation, 220 ES cell (see Embryonic stem cell) F Fate mapping (see Recombinase-based fate mapping) Flow cytometry, cell cycle analysis, 30–32 protein-fragment complementation assay, 454 G Gap junction (see Connexin) Gene chip array (see also Complementary DNA array hybridization) complexity of developmental genes, 269 gene trapping applications, 363, 364 tools in developmental genetics, 269–271 transcription factor target identification, cell lines, generation, 275, 276 inducible gene expression, 276, 277 overview, 274, 275 hybridization of chips with biotinylated RNA, first strand synthesis, 277 hybridization solution and conditions, 279 probe quantification and fragmentation, 279 RNA clean-up, 278 scanning and analysis, 280 second strand synthesis, 277, 278 staining, 279 transcription in vitro, 278 washing, 279 materials, 271, 272 Northern blot for target identification, 280, 281 prospects, 281 492 SV40 T gene utilization, 272–274 Gene trap (see also Protein-fragment complementation assay) applications, 335, 360–362 electroporation, 339, 366, 369, 370 embryonic stem cell clone picking, 339, 340, 343 embryo recovery, 367 feeder plates, 339, 342, 343 β-galactosidase staining, embryonic stem cell clones, 340, 368, 373 embryos, 342, 344, 368, 369, 373 fixation, 367, 368, 373 materials, 364, 365 genotyping of mice, adapter-mediated polymerase chain reaction, adapter ligation, 372 amplification reactions, 372 endonuclease digestion, 372 gel electrophoresis of products, 373 genomic DNA extraction, 372 materials, 366 principles, 359 breeding of homozygotes, 360 heterozygous mutants, 358 homozygotes, 358, 360 inverse polymerase chain reaction, 359, 366, 370, 371 plasmid rescue of trapped locus, 356, 358, 359 restriction fragment length polymorphisms, 358 materials, 337, 338, 364–367 media, 365, 366 morula aggregation, 342 mutagenicity of vectors, 356–358 overview, 335–337 phage libraries, 356 prospects, DNA chip, 363, 364 modification of trapped loci, 362 nuclear transfer, 362, 363 rationale, 347, 348 retroviral vector infection, 366, 370 screening of gene trap cell lines, 340, 343 strategies, enhancer trap, 348, 349 Index exon trap, 349 gene trap, 349, 350 induction strategies, 354, 355 reporter–selector cassettes, 351, 352 SA-type vectors, 350, 351, 353 SD-type vectors and poly (A) trap, 351, 353, 354 vector integration bias, 352, 353 supF complementation for gene identification, 356 tagged gene identification using 5'-RACE, rapid amplification, 341, 344 RNA extraction, 340, 341 overview, 355, 356 Glial-restricted precursor (GRP), isolation from embryonic stem cell cultures, differentiation conditions, 195 embryoid body formation, 191, 194, 195, 201, 202 feeder layer culture, 190, 192, 193, 201, 202 immunocytochemistry, cell surface, 200, 203 double and triple labeling, 201, 203 fluorescence cytoplasmic stainings, 200, 203 horseradish peroxidase, 200, 201, 203 materials, 192, 202 isolation, immunopanning, 191, 195, 196, 199, 203 magnetic bead sorting, 191, 196, 197, 199 overview, 197–199 materials, 190–192 maturation of precursor cells, 191, 192, 197, 199, 200 overview, 189, 190 undifferentiated embryonic stem cell culture, 190, 193, 194, 201 isolation from sox2-targeted marker cells, differentiation and culture, 208, 210, 211, 213, 214 efficiency of selection, 206, 207 embryonic stem cell maintenance, 207–209, 213 Index β-galactosidase staining, 208, 209, 211 immunocytochemistry of neural markers, 209, 211, 214 intra-uterine injection into fetal brain, 209, 211–214 materials, 207–209, 213 overview, 205, 206 substrate-coated plastic preparation, 209, 210 markers in differentiation, 205, 206 GRP (see Glial-restricted precursor) H Hair follicle cell, embryonic stem cell differentiation, Ayoub Shklar staining for keratin, 256, 259 embryoid body culture, 257 embryonic stem cell culture and maintenance, 256, 257 immunofluorescence of differentiating cells, 257, 259 materials, 255, 256, 259 formation overview, 255 Hematopoietic cell, embryonic stem cell differentiation, induction, culture, 76–79 materials, 74, 75, 77 principles, 73, 74 overview, 72 tetracycline-regulated gene expression analysis of hematopoiesis, Bcr-Abl clone establishment, 89, 90, 93 differentiation induction, 86, 90–93 embryonic stem cell maintenance, 85–88, 93 materials, 85, 86, 93 OP9 stromal cell line maintenance, 86, 90, 93 overview, 83–85 Tet-off parental cell line generation, 88, 89 vectors, 86, 87 Hsp27, embryonic stem cell modification for under/overexpression, antisense knockdown, clone selection, 40 493 transfection of CGR8 cells, 39–41 cell culture, 38 cell cycle analysis, 41, 42 cell death analysis, 42, 44 differentiation induction, 38, 39 differentiation marker detection with reverse transcription-polymerase chain reaction, 39 Hsp27 expression analysis with blots, 39, 44 materials, 36–38 expression in development, 35, 36 function, 35 Hypoxia, embryoid body, colony-forming unit scoring, 60, 61 culture from stem cells, 58–61 disaggregation and replating of progenitors, 59–61 methylcellulose for differentiation and replating, 57 oxygen gradients, 55, 56 embryonic stem cells culture for study, gelatin-adapting, 58, 60 hypoxic environment, 56 media, 56, 57 thawing, 58 hypoxia-inducible factor, 55 uterine conditions, 55 K Keratin, Ayoub Shklar staining, 233, 248, 256, 259 L β-Lactamase (see Protein-fragment complementation assay) M Macroarray hybridization, complementary DNA clone preparation, 427, 428 data analysis, 429–431 hybridization conditions, 429, 430 inserts amplification, 428, 430, 431 materials, 426, 427, 430 overview, 425, 426 RNA isolation, 426, 427, 430 spotting, 428 target, preparation, 428, 429, 431 494 purification, 429 Melanocyte, embryonic stem cell differentiation, counting of melanocytes, 265, 266 embryonic stem cell maintenance, 264, 266 induction, 265–267 markers, 262 materials, 262–264, 266 overview, 261, 262 ST2 stromal cell feeder layer preparation, 264–266 neural crest origin, 261 Membrane array (see Complementary DNA array hybridization) Mesodermal cell, embryonic stem cell differentiation, induction, culture, 75, 77 materials, 74, 77 principles, 72 overview, 71, 72 N National Center for Biotechnology Information (NCBI), gene bank growth, 347 NCBI (see National Center for Biotechnology Information) Neuronal-restricted precursor (NRP) (see also Stromal cell-derived inducing activity) isolation from embryonic stem cell cultures, differentiation conditions, 195 embryoid body formation, 191, 194, 195, 201, 202 feeder layer culture, 190, 192, 193, 201, 202 immunocytochemistry, cell surface, 200, 203 double and triple labeling, 201, 203 fluorescence cytoplasmic stainings, 200, 203 horseradish peroxidase, 200, 201, 203 materials, 192, 202 immunopanning, 191, 195, 196, 201, 202 materials, 190–192 maturation of precursor cells, 191, 192 Index overview, 189, 190 undifferentiated embryonic stem cell culture, 190, 193, 194, 201 isolation from sox2-targeted marker cells, differentiation and culture, 208, 210, 211, 213, 214 efficiency of selection, 206, 207 embryonic stem cell maintenance, 207–209, 213 β-galactosidase staining, 208, 209, 211 immunocytochemistry of neural markers, 209, 211, 214 intra-uterine injection into fetal brain, 209, 211–214 materials, 207–209, 213 overview, 205, 206 substrate-coated plastic preparation, 209, 210 markers in differentiation, 205, 206 NRP (see Neuronal-restricted precursor) O Oct3/4, embryonic stem cell differentiation role, 45 Osteoclast, cytokine control, 97 embryonic stem cell induction of differentiation, embryonic stem cell maintenance, 100, 101, 104 materials, 99, 100, 103, 104 multistep culture on OP9 cells, 102–105 OP9 stromal cell maintenance, 101, 104 overview, 97, 98 single-step culture, 101, 102, 104 ST2 stromal cell maintenance, 101, 104 markers, 97 single-cell polymerase chain reaction of osteoprogenitors, 403, 404, 407, 408 tartrate-resistant acid phosphatase staining, 103, 105 P p53, cell cycle pathway, 28, 29 Parkinson's disease, stromal cell-derived inducing activity neurons and grafting application, 221–223 Pax6, embryonic stem cell differentiation marker, 22, 23 Index PCA (see Protein-fragment complementation assay) PCR (see Polymerase chain reaction) Pem, embryonic stem cell modification for under/overexpression, characterization of embryoid bodies, cell mixing experiments, 50, 51 growth, 49, 50 morphology, 50 tissue-specific gene expression, 51 clone isolation, 48, 49, 52, 53 materials, 46, 47 principles, 46 teratoma analysis, 52 transfection, 48, 52 vector construction, 47, 52 expression in development, 45 Phage display, antibodies, amplification, 384, 390, 391 applications, 381 cell phage selection, 386 clone identification, 382 coated protein phage selection, 386, 391 concentration of unselected phage, 384, 385 enzyme-linked immunosorbent assay, 387, 391 expression of single-chain antibody, baculovirus expression, 389, 391 Escherichia coli expression, 388–391 growth of selected libraries, 386, 387, 391 materials, 382–384, 390 naive libraries, 382 peptide bead phage selection, 385, 391 problems, 381 purification of antibodies, 389–391 screening of individual clones, 387, 388, 391 single chain Fv (see Antibody) applications, 461 direct selection of complementary DNAs, coat protein selection for display, 461, 462 complexity testing, 465, 467 insert preparation, 464, 466 495 library generation, 464–466 materials, 462, 463 phage enrichment with affinity for ligand, 465–468 vector preparation, 463, 464, 466 gene trapping libraries, 356 phagemids, 381, 390 Phage-mediated gene transfer, advantages, 394 conferred tropism, 393, 394 DNA strand conversion, 395, 396, 399 efficiency, 393, 394 endotoxin removal, 398, 400 genotoxic treatments, 397–399 materials, 396, 397 phage concentration from cultured media, 397, 399, 400 reporter genes, 393 targeting ligand identification, 395, 399 titering, 398, 400 transfection, 396–398 transformation of bacteria, 397 vectors, 396 Polymerase chain reaction (PCR), gene trapping, adapter-mediated polymerase chain reaction, adapter ligation, 372 amplification reactions, 372 endonuclease digestion, 372 gel electrophoresis of products, 373 genomic DNA extraction, 371, 372 materials, 366 principles, 359 inverse polymerase chain reaction, 359, 366, 370, 371 muscle cell transfectant analysis, 131, 132, 146, 151 R 5'-RACE (see Gene trap) recombinase-based fate mapping, 318, 319, 326–328 RNA (see Reverse transcription polymerase chain reaction) single-cell polymerase chain reaction, cell isolation, materials, 404–406, 412 overview, 408 replica plating, 409, 410, 413 496 single cells and colonies, 408, 409, 412 complementary DNA fingerprinting, arbitrarily primed polymerase chain reaction, 411, 413 expressed sequence tag identification, 411, 412, 414 materials, 407 overview, 410, 411 osteoprogenitors, 403, 404, 407, 408 poly (A) polymerase chain reaction, advantages, 407, 408 amplification reactions, 410, 413 materials, 406, 407, 412 single chain Fv genes, 438, 443, 444 Protein-fragment complementation assay (PCA), advantages, 447, 448 applications, 449, 450, 451 dihydrofolate reductase system, fluorescence assay, flow cytometry, 453, 454 fluorescence microscopy, 453, 456, 457 fluorometer analysis, 454 materials, 451, 452 overview, 449, 450, 451 survival assay, 453, 456 β-lactamase system, colorimetric assay, 454, 457 enzymatic assay and fluorescence microscopy, 454, 455, 457 materials, 452 overview, 449, 450, 451 principles, 448, 449 Protein–protein interactions (see Phage display; Protein-fragment complementation assay; Yeast two-hybrid system) R 5'-RACE (see Gene trap) Rb (see Retinoblastoma protein) Recombinase-based fate mapping, indicator mice, expression profiling, β-galactosidase reporter detection, 319, 328 importance, 313, 314 materials, 318, 319 Index polymerase chain reaction genotyping, 318, 319, 327, 328 tail biopsy, 318, 326 germ line transmission of transgene, 318, 326 promoter selection, 312, 313 reporter selection, 313 map generation, alkaline phosphatase reporter staining, 329–330 double transgenic embryo identification with polymerase chain reaction genotyping, 328 materials, 319 principles, 309, 310 recombinase expression detection, in situ hybridization, gelvatol mounting media preparation, 317, 324, 330 hybridization incubations, 321–324, 330 materials, 315–317, 329 riboprobe synthesis, 316, 320, 321, 329, 330 tissue pretreatment and cryosectioning, 315, 320 β-galactosidase reporter detection, histochemistry, 325, 330 immunohistochemistry, 325 materials, 317, 318 recombinase-expressing mice, 310–312 recombination systems, Cre/LoxP, 285, 286, 309 Flp, 285, 286, 309 Flp variants, 314, 315 streptomyces phage integrase, 285, 286, 15 Retinoblastoma protein (Rb), cell cycle pathway, 27, 28 Reverse transcription-polymerase chain reaction (RT-PCR), activin, 22 adipocyte differentiation analysis, annealing temperature, 113, 114 primers, 110 RNA preparation, 110, 113, 115 bone morphogenetic proteins, 23 cardiomyocyte differentiation analysis, amplification reactions, 138, 150 cell sample preparation, 137, 149 Index gel electrophoresis of products, 139 materials, 129, 130, 149 primers and genes, 140 reverse transcription, 138, 150 RNA isolation, 137, 138, 150 connexin expression, amplification reactions, cDNA synthesis, materials, 129, 130, 149 RNA extraction, 65, 66 epidermal stem cell differentiation, amplification reactions, 250 DNA removal, 249 materials, 234 primers, 250, 251 reverse transcription, 250 RNA extraction, 248, 249 Pax6, 22, 23 Rex-1, 22 skeletal muscle cell differentiation analysis, amplification reactions, 138, 150 cell sample preparation, 137, 149 gel electrophoresis of products, 139 materials, 129, 130, 149 primers and genes, 140 reverse transcription, 138, 150 RNA isolation, 137, 138, 150 vascular smooth muscle cell differentiation analysis, amplification reactions, 138, 150 cell sample preparation, 137, 149 gel electrophoresis of products, 139 materials, 129, 130, 149 primers and genes, 140 reverse transcription, 138, 150 RNA isolation, 137, 138, 150 Rex-1, embryonic stem cell differentiation marker, 22 RT-PCR (see Reverse transcriptionpolymerase chain reaction) S SDIA (see Stromal cell-derived inducing activity) Skeletal muscle cell, embryonic stem cell differentiation, culture, cell lines, 128 feeder layers, 133 media, 128, 129, 149 undifferentiated embryonic stem cells, 134, 149 497 differentiation culture, 134, 136, 149 equipment, 132, 133 factors affecting, 128 immunofluorescence analysis, 130, 139, 141, 150 overview, 127 reverse transcription-polymerase chain reaction, amplification reactions, 138, 150 cell sample preparation, 137, 149 gel electrophoresis of products, 139 materials, 129, 130, 149 primers and genes, 140 reverse transcription, 138, 150 RNA isolation, 137, 138, 150 transfection, analysis, DNA isolation, 131, 145, 146 polymerase chain reaction, 131, 132, 146, 151 Southern blot, 132, 148, 151 clone selection, 145, 150 DNA preparation, stable transfections, 143, 150, 151 transient transfection, 143, 150, 151 electroporation, 143–145, 150 gain-of-function and loss-of-function analysis, 148, 149 materials, 131 rationale for differentiation studies, 141, 143 Stromal cell-derived inducing activity (SDIA), advantages, 220, 221 bone morphogenetic protein neural induction in ectoderm, 217, 218 cell lines, 223 coculture systems, 218 dopaminergic neuron induction, 219 embryonic stem cells, differentiation induction, 224, 225 mitomycin C treatment, 224, 226 epidermal differentiation induction, 220 grafting of induced neurons and Parkinson's disease application, 221–223 isolation of differentiated neural cells from feeder layer, 224, 226 materials, 223–225 nestin marker detection, 218, 219 neural induction mechanisms, 221 498 PA6 cell maintenance and feeder layer preparation, 223, 225 T Tartrate-resistant acid phosphatase (TRAP), osteoclast staining, 103, 105 Transgenic mouse, conditional transgenesis mediated by embryonic stem cells, cell lines, 289 DNA isolation, 292, 301 drug-resistant clone growth, 298–300, 305 embryonic fibroblast feeders, 289, 290 embryonic stem cell introduction into mice, aggregation with cleavage stage embryos, 302, 303, 305 cell preparation for aggregation, 301, 302, 305 embryo recovery, 302 tetraploid embryo generation, 303, 304, 306 equipment, 288, 289, 293 freezing and thawing of embryonic stem cells, 296, 297, 300 β-galactosidase staining, 292, 293, 300, 305 gene transfer in embryonic stem cells, electroporation, 297, 298 lipofection, 298, 305 media, 289, 292, 294–296, 304, 305 mitomycin C treatment of feeders, 291 overview, 287, 288 passage of cells, 294, 295, 305 preimplantation embryo culture, 293, 294 fate mapping (see Recombinase-based fate mapping) gene trapping (see Gene trap) knockout mouse limitations in functional analysis, 348 recombination systems, Cre/LoxP, 285, 286, 309 Flp, 285, 286, 309 inducible systems, 286 reporter testing, 287 streptomyces phage integrase, 285, 286 TRAP (see Tartrate-resistant acid phosphatase) Index Two-hybrid system (see Yeast two-hybrid system) V Vascular smooth muscle cell, embryonic stem cell differentiation, culture, cell lines, 128 feeder layers, 133 media, 128, 129, 149 undifferentiated embryonic stem cells, 134, 149 differentiation culture, 134, 136, 137, 149 equipment, 132, 133 factors affecting, 128 immunofluorescence analysis, 130, 139, 141, 150 overview, 127 reverse transcription-polymerase chain reaction, amplification reactions, 138, 150 cell sample preparation, 137, 149 gel electrophoresis of products, 139 materials, 129, 130, 149 primers and genes, 140 reverse transcription, 138, 150 RNA isolation, 137, 138, 150 transfection, analysis, DNA isolation, 131, 145, 146 polymerase chain reaction, 131, 132, 146, 151 Southern blot, 132, 148, 151 clone selection, 145, 150 DNA preparation, stable transfections, 143, 150, 151 transient transfection, 143, 150, 151 electroporation, 143–145, 150 gain-of-function and loss-of-function analysis, 148, 149 materials, 131 rationale for differentiation studies, 141, 143 Y Yeast two-hybrid system, antigen–intracellular single chain Fv interaction screening, 436–438, 441–443, 445 autoactivation testing of reporter genes, Index GAL1-HIS3, 478, 484 lacZ, 478, 481 bacterial strains, 473 deletion mapping of interactive domains, 483 fusion plasmid preparation, 476 fusion protein expression, 478, 479 library screening, 480, 481 library transformation efficiency test, 479, 480, 484 limitations, 447 media, 474, 475 499 plasmids, 473, 476 positive samples, characterization, 482 false positives, 484 reconstruction, 482, 483 storage, 481, 482 strength of interaction characterization, 483 principles, 471, 472 solutions, 475, 476 transformation, 471, 472, 476–478, 484 yeast strains, 471, 472 METHODS IN MOLECULAR BIOLOGY • 185 TM Series Editor: John M Walker Embryonic Stem Cells Methods and Protocols Edited by Kursad Turksen Ottawa Health Research Institute, Ottawa, Ontario, Canada Embryonic stem (ES) cells have significant potential in basic studies designed to better understand how different cells and tissues in the body are formed, as well as for generating unlimited numbers of cells for transplantation, drug delivery, and drug testing In Embryonic Stem Cells: Methods and Protocols, Kursad Turksen and a panel of international experts describe their most productive methods for using ES cells as in vitro developmental models for many cell and tissue types Set out in step-by-step detail by the investigators who developed them, these protocols range widely from ES cell isolation, maintenance, and modulation of gene expression, to cutting-edge techniques that use cDNA arrays in gene expression analysis and phage display libraries There are also advanced techniques for the generation of antibodies against very rare antigens and for the identification and characterization of proteins and protein interactions Additional studies of the ES cell cycle and apoptosis, as well as protocols for the use of ES cells to generate diverse cell and tissue types, complete this collection of readily reproducible methods Many of the techniques have already been shown to have tremendous utility with ES cells and their differentiated progeny Authoritative and state-of-the-art, this unique first collection of protocols for the study of ES cells, Embryonic Stem Cells: Methods and Protocols, will prove an invaluable resource not only for those generally interested in cell and developmental biology, but also for those actively using, or planning to use, ES cells to study fate choices and specific lineages FEATURES • Detailed protocols for establishing and characterizing various lineages using ES cells • Reproducible protocols for the cellular and molecular manipulation of differentiating ES cells • Cutting-edge techniques for use with ES cells and other cell models of differentiation and development • Numerous tips from the experts to avoid pitfalls and ensure robust results CONTENTS Methods for the Isolation and Maintenance of Murine Embryonic Stem Cells The Use of Chemically Defined Media for the Analyses of Early Development in ES Cells and Mouse Embryos Analysis of the Cell Cycle in Mouse Embryonic Stem Cells Murine Embryonic Stem Cells as a Model for Stress Proteins and Apoptosis During Differentiation Effects of Altered Gene Expression on ES Cell Differentiation.Hypoxic Gene Regulation in Differentiating ES Cells Regulation of Gap Junction Protein (Connexin) Genes and Function in Differentiating ES Cells Embryonic Stem Cell Differentiation as a Model to Study Hematopoietic and Endothelial Cell Development Analysis of Bcr-Abl Function Using an In Vitro Embryonic Stem Cell Differentiation System Embryonic Stem Cells as a Model for Studying Osteoclast Lineage Development Differentiation of Embryonic Stem Cells as a Model to Study Gene Function During the Development of Adipose Cells Embryonic Stem Cell Differentiation and the Vascular Lineage Embryonic Stem Cells as a Model to Study Cardiac, Skeletal Muscle, and Vascular Smooth Muscle Cell Differentiation Cardiomyocyte Enrichment in Differentiating ES Cell Cultures: Strategies and Applications Embryonic Stem Cells as a Model for the Physiological Analysis of the Cardiovascular System Isolation of Lineage-Restricted Neural Precursors from Cultured ES Cells Lineage Selection for Generation and Amplification of Neural Precursor Cells Selective Neural Induction from ES Cells by Stromal Cell-Derived Inducing Activity and Its Potential Therapeutic Application in Parkinson’s Disease Epidermal Lineage ES Cell Differentiation Into the Hair Methods in Molecular BiologyTM • 185 EMBRYONIC STEM CELLS, METHODS AND PROTOCOLS ISBN: 0-89603-881-5 Follicle Lineage In Vitro Embryonic Stem Cells as a Model for Studying Melanocyte Development Using Progenitor Cells and Gene Chips to Define Genetic Pathways ES Cell-Mediated Conditional Transgenesis Switching on Lineage Tracers Using Site-Specific Recombination From ES Cells to Mice: The Gene Trap Approach Functional Genomics by Gene-Trapping in Embryonic Stem Cells Phage-Displayed Antibodies to Detect Cell Markers Gene Transfer Using Targeted Filamentous Bacteriophage Single-Cell PCR Methods for Studying Stem Cells and Progenitors Nonradioactive Labeling and Detection of mRNAs Hybridized onto Nucleic Acid cDNA Arrays Expression Profiling Using Quantitative Hybridization on Macroarrays Isolation of Antigen-Specific Intracellular Antibody Fragments as Single Chain Fv for Use in Mammalian Cells Detection and Visualization of Protein Interactions with Protein Fragment Complementation Assays Direct Selection of cDNAs by Phage Display Screening for Protein–Protein Interactions in the Yeast TwoHybrid System in Embryonic Stem Cells Index 90000 780896 038813 ... Publication Data Embryonic Stem Cells: methods and protocols / edited by Kursad Turksen p cm (Methods in molecular biology ; v 185) Includes bibliographical references and index ISBN 0-8 960 3-8 8 1-5 (alk... 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,... Volumes and Cell Counts for ES Cells in Various Different Tissue Culture and Multiwell Dishes Dish Size 4-well dish 24-well dish 12-well dish 6-well dish 35-mm dish 60-mm dish 100-mm dish 4-chamber