Contents

Preface v

Contributors xi

1 Nuclear Transplantation in Xenopus

John B. Gurdon 1

2 Nuclear Transfer in Sheep

WilliamA. Ritchie 11

3 Protocols for Nuclear Transfer in Mice

Shaorong Gao 25

4 Isolation of Stromal Stem Cells From Human Adipose Tissue Andrew C. Boquest, Aboulghassem Shahdadfar,

Jan E. Brinchmann, and Philippe Collas 35

5 Nuclear Reprogramming by Cell Fusion

Robert H. Broyles, Austin C. Roth, Mairead Todd, and Visar Belegu 47

6 Polyethylene Glycol-Mediated Cell Fusion

Jian Yang and Ming Hong Shen 59

7 Epigenetic Reprogramming of Somatic Genomes by Electrofusion With Embryonic Stem Cells Masako Tada and Takashi Tada 67

8 Quantification of Cell Fusion by Flow Cytometry Stephen Sullivan, Martin Waterfall, Ed J. Gallagher,

Jim McWhir, and Steve Pells 81

9 Modulation of Cell Fate Using Nuclear and Cytoplasmic Extracts Anne-Mari Hakelien, Kristine G. Gaustad, and Philippe Collas 99

10 Transgenic Systems in Nuclear Reprogramming

Megan Munsie, Peter Mountford, and Jennifer Nichols 115

11 Using Immunofluorescence to Observe Methylation Changes in Mammalian Preimplantation Embryos Fatima Santos and Wendy Dean 129

12 Observing S-Phase Dynamics of Histone Modifications

With Fluorescently Labeled Antibodies Rong Wu,Anna V. Terry, and David M. Gilbert 139

13 Quantitative Analysis of Telomerase Activity and Telomere

Length in Domestic Animal Clones Dean H. Betts,Steven Perrault,Lea Harrington, and W. Allan King 149

14 Pluripotency: Capacity for In Vitro Differentiation of Undifferentiated Embryonic Stem Cells Cornelia Wiese, Gabriela Kania, Alexandra Rolletschek,

Przemyslaw Blyszczuk, and Anna M. Wobus 181

15 Staining Embryonic Stem Cells Using Monoclonal Antibodies to Stage-Specific Embryonic Antigens Bruce A. Fenderson, Maria P. De Miguel, April D. Pyle, and Peter J. Donovan 207

16 Analysis of the Nucleolar Compartment of the Nucleus as an

Indicator of Nuclear Reprogramming After Nuclear Transfer Jacques-E. Flechon 225

17 Methylation-Sensitive Polymerase Chain Reaction Hannah R. Moore, Richard R. Meehan, and Lorraine E. Young 239

18 Analysis of DNA Methylation Profiles in Preimplantation Embryos

Using Bisulfite Mutagenesis Yong-Mahn Han, Seok-Ho Kim, and Yong-Kook Kang 251

19 Chromatin Immunoprecipitation Assay for Mammalian Tissues

Fiona B. Turner,Wang L. Cheung, and Peter Cheung 261

20 Histone Modifications and Transcription Factor Binding on Chromatin: ChlP-PCR Assays Jaejoon Won and Tae Kook Kim 273

21 In Vivo Genomic Footprinting Using LM-PCR Methods

Hiromi Tagoh, Peter N . Cockerill, and Constanze Bonifer 285

22 Analyzing Histone Modification Using Crosslinked Chromatin

Treated With Micrococcal Nuclease Pascal Lefevre and Constanze Bonifer 315

Index 327

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