Experimental Study On The Construction Of Cloned Embryos Through Human-rabbit Inter-species Somatic Nuclear Transfer

Objective: In order to pave the way for therapeutic cloning, inter-species cloned embryos were reconstructed with human somatic cell as nuclear donor and enucleated rabbit oocyte as recipient. Also, the effects of type, passage and preparation method of donor cells on development of embryos were studied.Methods: At the first stage, three kinds of in vitro cultured somatic cells(ossocartilaginous cells, fibroblasts and granulose cells) were microinjected into the subzonal of enucleated M II rabbit oocytes to obtain reconstructed eggs. After electrofusion, stimulation and in vitro culture, the development of embryos reconstructed by different type, different passage and different preparation method were compared. At the second stage, FISH were conducted to detect nuclear deriviation of blastomere.Results: The number of reconstructed eggs obtained in our experiment with osseocartilaginous cells, granulose cells and fibroblast used as donor cell were 67,116 and 178 respectively; their electrofusion rate were 61.2%, 56.9% and 55.6% respectively, showing no significant difference. The cleavage rate of three groups were 39%,64.2% and 42.2%, while the 8-cell embryo rate were 19.5%,53.0% and 25.3% respectively, showing that the early development rate of the granulose cells were significant higher than the other two(P<0.05 ). There are embryos in all these three groups which can develop to morula stage, the rate were 7.3%, 31.8% and 12.1% respectively; with the granulose cell groupshow significantly higher rate than the others (P<0.01 ). However, only embryos from the granulose cell group and the fibroblast group can develop to blastocyst with the rate being 18.2% and 6.1%, the blastocyst rate of granulose cell group was significantly higher than the fibroblast group. No blastocyst was obtained in osseocartilaginous cell group in our experiment. Study on the effect of passages on development competency of cloned embryos showed that: the eletrofusion rates were not significant different among these three groups; the early development rate of the reconstructed embryos from the second passage was significantly higher than the other two; except the third passage, the reconstructed embryos from the other two passages can develop to blastocyst, and the difference between their development rate was not significant. Research on the effect of donor cell preparation method to nuclear transfer efficiency showed that: the early development rate of cloned embryos in each group showed no significant difference. None of the embryos reconstructed by fresh fibroblast and fibroblast chilled for 9 days develope to blastocyst while parts of embryo in the other two groups can develop to blastocyst without significant difference in rate. Detection showed that the genome of the inter-species cloned embryos reconstructed in our experiment came from human.Conclusion: Cloned embryos can be constructed through human- rabbit inter-species nuclear transfer; different kinds of somatic cell result in different efficiency of nuclear transfer, while the in vitro passage of donor won't influence embryo development; meanwhile, refrigeration is a convenient and efficient donor cell preparation methods. It's feasible to detect DNA genotype through FISH.