Application of Cultural System Improved in Mouse Somatic Cell Nuclear Transfer and Isolation of ES Cells from Reconstructed Embryos

Female mice after 8 weeks were superovulated with PMSG and hCG, and their eggs were taken as recipients. Meanwhile cumulus cells around oocytes were also collected and used as donor cells in somatic cell nuclear transfer. Oocytes injected with donor cell nuclei were activated for 6 h by treatment in M16 media modified containing SrCl2, and then cocultured with mouse oviduct epithelial cell in mM16 medium. When being in the stage of blastocyst, they were transferred on the feeder layers of mouse embryonic fibroblasts, adding ES cell media conditioned. After hatched from blastocysts, ICM were isolated and trypsinized, and then cocultured continuously to gain ES cell masses. Results indicated that activation rate of embryos reconstructed was 65.23%, development rate of blastocyst was 11.69%; ES cell colonies were isolated from 9 blastocysts reconstructed, isolation rate was 2.77%. ES cell colonies isolated were with islandlike images and strong positive by AKP staining, could become embryo bodies and spontaneously differentiate into epidermal-like cells around them in vitro. In addition, after frozen and thawed routinely, ES cell colonies were with strong proliferation and previous image. It indicates that mouse oviduct epithelial cell, modified M16 media and ES cell media with cardiomyocyte media can be more successfully applied in mouse somatic cell nuclear transfer and isolation of ES cell from reconstructed embryos.