Improving The Efficiency Of Bovine Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer(SCNT) technique which can be used to breed superior domestic animals ,product experimental animals, survive endangered mammalian species, therapeutical cloning and obtain transgenic animals will have a bright prospect in application. However, the low efficiency of SCNT restricts its development. Since the cloning mechanism have not be known clearly, the improvement of some key steps of this technique will provide an effective way to increase the NT efficiency. In this study, Oocytes recovered by ovum pick up (OPU) were used as recipient cytoplasm for somatic nuclear transfer (SCNT). In the present study, we compared some critical steps in bovine SCNT procedure including enucleation mode, treatment of donor cell, activation method, co-culture system. Our results shown that the enucleation with the help of Hoechst33342 staining and UV light could enucleate completely(100.00%) compared with"blind enucleation"(58.52%). Pronase protease made the donor cells stick to the oocyte membrane tightly, lending to higher fusion rate than that of the group without the treatment of pronase protease (78.69% versus 54.88%). Although chemical activation and electrical activation resulted in a similar cleavage (59.35% versus 58.84%), the death after activation of electrical activation was higher than that of chemical activation (23.26% versus 5.10%). ACM(culture medium)+ mouse embryonic fibroblast(feeder cell) co-culture system resulted in a higher blastocyst and 90-d pregnancy than that of B2(culture medium)+cumulus cell(feeder cell) co-culture system (blastocyst:44.91% versus 35.24%;pregnancy:35.37% versus 9.26%). In conclusion enucleation with the help of Hoechst33342 staining and UV light +chemical activation +ACM(culture medium)+ mouse embryonic fibroblast(feeder cell) co-culture system could improve the bovine SCNT efficiency significantly.