Nuclear Reprogramming Methods and Protocols

About this book

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.

Contents

Preface; Contributors; 1. Nuclear Transplantation In Xenopus; J B Gurdon; 2. Nuclear Transfer in the sheep; William A. Ritchie; 3. Protocols For Nuclear Transfer In The Mouse; Shaorong Gao; 4. Isolation of Stromal Stem Cells from Human Adipose Tissue; Andrew C. Boquest, Aboulghassem Shahdadfar, Jan E. Brinchmann and Philippe Collas; 5. Nuclear Reprogramming by Cell Fusion; Robert H. Broyles, Austin C. Roth, Mairead Todd and Visar Belegu; 6. Polyethylene glycol (PEG) mediated cell fusion; Jian Yang and Ming Hong Shen; 7. Epigenetic reprogramming of somatic genomes by electrofusion with embryonic stem cells; Masako Tada and Takashi Tada; 8. Quantification of Cell Fusion by Flow Cytometry; Stephen Sullivan, Martin Waterfall, Jim McWhir and Steve Pells; 9. Modulation of Cell Fate Using Nuclear and Cytoplasmic Extracts; Anne-Mari Hakelien, Kristine G. Gaustad and Philippe Collas; 10. Transgenic Systems in Nuclear Reprogramming; Megan Munsie, Peter Mountford and Jennifer Nichols; 11. Using immunofluorescence to observe methylation changes in mammalian preimplantation embryos; Fatima Santos and Wendy Dean; 12. Observing nuclear structure/chromatin changes with fluorescently-labelled antibodies; Rong Wu, Anna V. Terry and David M. Gilbert; 13. Quantitative Analysis of Telomerase Activity and Telomere Length in Domestic Animal Clones; Dean H. Betts, Steven Perrault, Lea Harrington, W. Allan King; 14. Pluripotency: Capacity for In Vitro Differentiation of Undifferentiated Embryonic Stem (ES) Cells; Cornelia Wiese, Gabriela Kania, Alexandra Rolletschek, Przemyslaw Blyszczuk and Anna M. Wobus; 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; 16. Analysis of the Nucleolar Compartment of the Nucleus as an Indicator of Nuclear Reprogramming after Nuclear Transfer; Jacques-E. Flechon; 17. Methylation-Sensitive PCR; Hannah R. Moore, Richard R. Meehan and Lorraine E. Young; 18. Analysis of DNA Methylation Profiles in Preimplantation Embryos Using Bisulfite Mutagenesis; Yong-Mahn Han, Seok-Ho Kim and Yong-Kook Kang. 19. Chromatin Immunoprecipitation Assay for Mammalian Tissues; Fiona B. Turner, Wang L. Cheung and Peter Cheung; 20. Histone Modifications and Transcription Factor Binding on Chromatin: ChIP-PCR Assays; Jaejoon Won and Tae Kook Kim; 21. In Vivo Genomic Footprinting Using LM-PCR Methods; Hiromi Tagoh, Peter N. Cockerill and Constanze Bonifer; 22. Analyzing Histone Modification Using Crosslinked Chromatin treated with Micrococcal Nuclease; Pascal Lefevre and Constanze Bonifer; Index.

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