Nuclear Reprogramming: Methods and ProtocolsSteve Pells 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
| 2 | |
John B Gurdon 2 Nuclear Transfer in Sheep 1 | 11 |
Protocols for Nuclear Transfer in Mice | 25 |
Isolation of Stromal Stem Cells From Human Adipose Tissue | 35 |
Nuclear Reprogramming by Cell Fusion | 47 |
Jian Yang and Ming Hong Shen Epigenetic Reprogramming of Somatic Genomes | 59 |
by Electrofusion With Embryonic Stem Cells | 67 |
Stephen Sullivan Martin Waterfall Ed J Gallagher | 81 |
With Fluorescently Labeled Antibodies | 139 |
Length in Domestic Animal Clones | 149 |
Capacity for In Vitro Differentiation | 181 |
Bruce A Fenderson Maria P De Miguel April D Pyle | 207 |
Analysis of the Nucleolar Compartment of the Nucleus as | 225 |
MethylationSensitive Polymerase Chain Reaction | 239 |
Analysis of DNA Methylation Profiles in Preimplantation Embryos | 251 |
In Vivo Genomic Footprinting Using LMPCR Methods | 285 |
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Common terms and phrases
acid agarose aliquots amplification analysis antigens approx assay Biol blastocyst bovine cell fusion cell line cell types centrifuge chromatin chromatin immunoprecipitation chromosomes cloned cover slips crosslinking culture dish cumulus cells cycle cytoplasm differentiation digestion diluted DNA methylation donor cell EDTA electrofusion embryonic stem cells embryos enucleation enzyme ethanol extract fibroblasts final concentration flow cytometry fluorescence gene genomic DNA human hybrid cells immunoprecipitation Incubate injection lysis buffer membrane Methods and Protocols mg/mL microscope MNase Molecular mouse NaCl Note Nuclear Reprogramming nuclear transfer nuclei nucleosome oocytes PCR products pellet permeabilized pipet plate pluripotent Prepare primer protein reaction room temperature sample secondary antibody sequence serum Sigma slide sodium solution somatic cell staining sterile stored at 20°C stromal stem cells Subheading supernatant suspension telomerase telomerase activity telomere telomere length thymocytes tion Tris-HCl tube vitro vivo Wash µg/mL