Nuclear Reprogramming: Methods and Protocols / Edition 1

by Steve Pells

Overview

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.

Product Details

ISBN-13:	9781588293794
Publisher:	Springer-Verlag New York, LLC
Publication date:	12/05/2005
Series:	Methods in Molecular Biology , #325
Edition description:	2006
Pages:	334
Product dimensions:	6.10(w) x 9.25(h) x 0.04(d)

Table of Contents

Nuclear Transplantation in Xenopus.- Nuclear Transfer in Sheep.- Prools for Nuclear Transfer in Mice.- Isolation of Stromal Stem Cells From Human Adipose Tissue.- Nuclear Reprogramming by Cell Fusion.- Polyethylene Glycol-Mediated Cell Fusion.- Epigenetic Reprogramming of Somatic Genomes by Electrofusion With Embryonic Stem Cells.- Quantification of Cell Fusion by Flow Cytometry.- Modulation of Cell Fate Using Nuclear and Cytoplasmic Extracts.- Transgenic Systems in Nuclear Reprogramming.- Using Immunofluorescence to Observe Methylation Changes in Mammalian Preimplantation Embryos.- Observing S-Phase Dynamics of Histone Modifications With Fluorescently Labeled Antibodies.- Quantitative Analysis of Telomerase Activity and Telomere Length in Domestic Animal Clones.- Pluripotency: Capacity for In Vitro Differentiation of Undifferentiated Embryonic Stem Cells.- Staining Embryonic Stem Cells Using Monoclonal Antibodies to Stage-Specific Embryonic Antigens.- Analysis of the Nucleolar Compartment of the Nucleus as an Indicator of Nuclear Reprogramming After Nuclear Transfer.- Methylation-Sensitive Polymerase Chain Reaction.- Analysis of DNA Methylation Profiles in Preimplantation Embryos Using Bisulfite Mutagenesis.- Chromatin Immunoprecipitation Assay for Mammalian Tissues.- Histone Modifications and Transcription Factor Binding on Chromatin: ChIP–PCR Assays.- In Vivo Genomic Footprinting Using LM–PCR Methods.- Analyzing Histone Modification Using Crosslinked Chromatin Treated With Micrococcal Nuclease.