Titlebook: Nuclear Reprogramming; Methods and Protocol Nathalie Beaujean,Hélène Jammes,Alice Jouneau Book 2015Latest edition Springer Science+Business

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Treatment of Donor Cell/Embryo with Different Approaches to Improve Development After Nuclear Transracticed technique. Thus, it is very important to find simple and reproducible methods for improving the success rate of SCNT. In this chapter, we will review our recent protocols, which seem to be the simplest and most reliable method to date to improve development of SCNT embryos.

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,Micro Chromatin Immunoprecipitation (μChIP) from Early Mammalian Embryos,io output, suitable for as few as 100 cells. This chapter provides a detailed protocol for μChIP from early mammalian embryos, also suitable for any sample of limited numbers of cells. Minor modifications of this optimized high signal to noise ChIP protocol make it a reliable tool for the use with any cell number (100–10.).

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1064-3745 results.Contains key notes and implementation advice from t.Nuclear Reprogramming: Methods and Protocols, Second Edition. includes not only classic methods to perform nuclear transfer in different species but also several techniques to assess the early and late development of the reconstructed embr

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Nuclear Transfer and Transgenesis in the Pig,available for this species. In this chapter, we introduce our routine workflow for the production of genetically engineered pigs, especially focused on the genetic modification of somatic donor cells, SCNT using in vitro matured oocytes, and laparoscopic embryo transfer.

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Analysis of Nuclear Reprogramming Following Nuclear Transfer to , Oocyte,ogramming..Here, it is first explained how to carry out transplantation of multiple mammalian cell nuclei to . oocytes. It is then described how to perform RT-qPCR, Western Blot, Chromatin Immunoprecipitation, and live imaging analysis to monitor transcriptional reprogramming of the nuclei transplanted to oocytes.

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Assessment of Cell Lineages and Cell Death in Blastocysts by Immunostaining, to look at cell position and to analyze and count the proportions between the different cell types. Thus after any kind of embryo manipulation such as nuclear transfer (NT), the analysis of the three cell lineages by immunofluorescence will provide criteria for good or poor development.

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Methylation of Specific Regions: Bisulfite-Sequencing at the Single Oocyte or 2-Cell Embryo Level,e thus developed a very efficient protocol for methylation studies on individual oocytes or cleavage-stage embryos. All the different steps were optimized, from DNA extraction, to limit DNA degradation and give a high success rate of bisulfite converted DNA, to amplification of the bisulfite modified DNA.

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,Embryonic Stem Cell–Somatic Cell Fusion and Postfusion Enucleation,sion of mouse tetraploid ES cells with somatic cells and enrichment of the resulting heterokaryons. We next describe the conditions for the differential removal of the ES cell nucleus, allowing for the recovery of somatic cells.

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Assessing the Quality of Donor Cells: Karyotyping Methods,cribe the classical protocols that are needed to perform complete cytogenetic analyses, i.e., G-banding to identify chromosome aberrations, followed by Fluorescent In Situ Hybridization (FISH) of specific probes for a more sensitive detection and precise identification of the rearrangement.