Effect Of Major Factors On Sheep Cumulus Cell Somatic Nuclear Transfer

Somatic nuclear transplantation (SNT) is a powerful technique which is benefit for animal breeding, protecting and saving the species of wildlife which are rare or near extinction, and it also plays an important role for the solution of many basic biological problems.Most cells contain the same set of genes and yet they are extremely diverse in appearance. They are all derived from a totipotent zygote generated after fertilization of an oocyte. These newly acquired cell fates are propagated by heritable epigenetic mechanisms . The successful reprogramming of a differentiated donor cell back to a totipotent stage, by somatic cell nuclear transfer (NT), has been demonstrated in several species. Although viable animals have been cloned by nuclear transfer from somatic cells in many kinds of animal, the cloning and survival rate of cloned animals is remain very low. Now, it is considered that incomplete reprogramming genome epigenetic is the main factor caused the low efficiency of the somatic nuclear transfer.In this study, we mainly analysed the optimum concentration of demecolcine for chemically assisted enucleation, the effects of DMSO and FBS on the development rate of sheep cumulus cell nuclear transferred embryos, and the effects of 5-aza-dC on the development rate of sheep cumulus cell nuclear transferred embryos.The optimum concentration of demecolcine for chemically assisted enucleation is 0.5μg/ml, the sheep oocytes treated with 0.5μg/ml demecolcine for 0.5h had the highest extrusion cones rate.Addition of 1% (v/v) DMSO to the activation medium significantly improved the fusion rate of cloned embryos (P<0.01), and improved the frequency of development to the blastocyst stage(P<0.05).Addition of 10% (v/v) FBS to the in vitro culture medium significantly improved the frequency of development to the blastocyst stage(P<0.01).Differentiated somatic cells and embryos cloned from somatic cells by NT have higher levels of DNA methylation than gametes and early embryos produced in vivo. Reducing DNA methylation in donor cells and NT embryos by treating them with the DNA methyl-transferase inhibitor (5-aza-2'-deoxycytidine; 5-aza-dC) may improve cloning efficiency of NT embryos by providing NT embryos with similar epigenetic characteristics as in vivo embryos.A range concentrations (0 to 0.02μM) of 5-aza-dC were used to treat sheep cumulus cells and we found that the blastocyst rate of cloned embryos reconstructed by cumulus cells treated with 0.01μM 5-aza-dC was extremely higher than that of other groups (P<0.01), and its cleavage rate and morula rate have no different with that of other groups (P>0.05).A range concentrations (0 to 0.02μM) of 5-aza-dC were also used to treat cloned embryos reconstructed by cumulus cells, and the developmental potential of cloned embryos was much better than other groups when the cloned embryos were treated with 0.01μM 5-aza-dC, for its blastocyst rate was extremely higher than that of other groups(P<0.01).However, the blastocyst rate of sheep cumulus cell nuclear transferred embryos from both the cumulus cells and the cloned embryos treated with 0.01μM 5-aza-dC has no different with that of the sheep cumulus cell nuclear transferred embryos from cumulus cells treated with 0.01μM 5-aza-dC, and it also has no different with that of sheep cumulus cell nuclear transferred embryos from cloned embryos treated with 0.01μM 5-aza-dC(P>0.05).