Nuclear Reprogramming

Nuclear Reprogramming : Methods and Protocols

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Description

A wide-ranging collection of readily reproducible methods for performing nuclear reprogramming by nuclear transfer in several different species, by fusion through both chemical treatment and electrically shocking cells, and by in vivo treatment of cells with cell extracts. Several methods of monitoring nuclear reprogramming are also presented, including the use of transgenic markers, activation of telomerase as an ES-specific marker, light and electron microscopic observation of structural changes in the nucleus, and verification of surface marker expression and the differentiation potential of stem cells. Biochemical methods are provided for the examination of chromatin protein modifications, nucleosomal footprinting, transcription factor binding, and the study of DNA methylation changes both at the specific locus level and at the level of the whole nucleus.
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Product details

  • Hardback | 334 pages
  • 158 x 232 x 26mm | 680.4g
  • Totowa, NJ, United States
  • English
  • 2006 ed.
  • XIV, 334 p.
  • 1588293793
  • 9781588293794

Back cover copy

Although nuclear reprogramming-the cellular process underlying cloning and the artificial generation of stem cells from somatic cells-may be considered a "black box" whose interior events are either completely unknown or at least imperfectly characterized, tantalizing clues about its operation are beginning to emerge. In Nuclear Reprogramming: Methods and Protocols, hands-on laboratory scientists describe readily reproducible methods for actually performing nuclear reprogramming by nuclear transfer in several different species, by fusion induced both chemically and electrically, and by the in vivo treatment of cells with cell extracts. Isolation of an adult stem cell type is also described. In addition, several methods of monitoring nuclear reprogramming are presented, including the use of transgenic markers, activation of telomerase as an ES-specific marker, light and electron microscopic observation of structural changes in the nucleus, and the verification of surface marker expression and differentiation potential of stem cells. Biochemical methods are provided for the examination of chromatin protein modifications, nucleosomal footprinting, transcription factor binding, and the study of DNA methylation changes both at the specific locus level and at the level of the whole nucleus. The protocols follow the successful Methods in Molecular Biology(TM) series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Wide-ranging and highly practical, Nuclear Reprogramming: Methods and Protocols significantly expands the repertoire of methods available to perform, study, and understand nuclear programming, not only for cloning, but also for reprogramming somatic cells to stem cells.
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Table of contents

Nuclear Transplantation in Xenopus
John B. Gurdon

Nuclear Transfer in Sheep
William A. Ritchie

Protocols for Nuclear Transfer in Mice
Shaorong Gao

Isolation of Stromal Stem Cells From Human Adipose Tissue
Andrew C. Boquest, Aboulghassem Shahdadfar, Jan E. Brinchmann, and Philippe Collas

Nuclear Reprogramming by Cell Fusion
Robert H. Broyles, Austin C. Roth, Mairead Todd, and Visar Belegu

Polyethylene Glycol-Mediated Cell Fusion
Jian Yang and Ming Hong Shen

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

Quantification of Cell Fusion by Flow Cytometry
Stephen Sullivan, Martin Waterfall, Ed J. Gallagher, Jim McWhir, and Steve Pells

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

Transgenic Systems in Nuclear Reprogramming
Megan Munsie, Peter Mountford, and Jennifer Nichols

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

Observing S-Phase Dynamics of Histone Modifications With Fluorescently Labeled Antibodies
Rong Wu, Anna V. Terry, and David M. Gilbert

Quantitative Analysis of Telomerase Activity and Telomere Length in Domestic Animal Clones
Dean H. Betts, Steven Perrault, Lea Harrington, and W. Allan King

Pluripotency: Capacity for In Vitro Differentiation of Undifferentiated Embryonic Stem Cells
Cornelia Wiese, Gabriela Kania, Alexandra Rolletschek, Przemyslaw Blyszczuk, and Anna M. Wobus

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

Analysis of the Nucleolar Compartment of the Nucleus as anIndicator of Nuclear Reprogramming After Nuclear Transfer
Jacques-E. Flechon

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

Analysis of DNA Methylation Profiles in Preimplantation Embryos Using Bisulfite Mutagenesis
Yong-Mahn Han, Seok-Ho Kim, and Yong-Kook Kang

Chromatin Immunoprecipitation Assay for Mammalian Tissues
Fiona B. Turner, Wang L. Cheung, and Peter Cheung

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

In Vivo Genomic Footprinting Using LM-PCR Methods
Hiromi Tagoh, Peter N. Cockerill, and Constanze Bonifer

Analyzing Histone Modification Using Crosslinked Chromatin Treated With Micrococcal Nuclease
Pascal Lefevre and Constanze Bonifer

Index
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