Study On The Mouse Embryo Cloning By The Somatic Cell Nuclear Transplantation

KunMing(KM) strain mouse and 615 strain mouse were used as experimental animals in this study.Somatic cell nuclear transplantation in operational procedure was initially established.The criticalital fators in somatic cell nuclear transplantation procedure were approached.The study of transgenic mouse was established as experiment foundation in future.1.Effect of different in vivo maturation times after HCG injection on in vitro development of reconstructed embryo.The result was that the in vivo maturation oocyte rate of 11h,12h,13h,14h groups after HCG injection were significantly higher than that of groups(P<0.05). In vivo maturing oocyte rate after HCG injection before 11h or after 14h were gradually decreased;The reconstructed blastulas were only observed in the groups of 11h,12h,13h,14h,15h after HCG injection,the reconstructed blastulas in the groups of 11h,12h after HCG injection were significantly higher than that of groups(P<0.05).2.Effect of different chemical agents and electric stimulus on in vitro development of reconstructed embryo.The result was that in vitro development of reconstructed embryos in SrCl₂ activation group was significantly higher than that of groups(P<0.05).Reconstructed morulas were only obtained in this SrCl₂ activation group.The in vitro development of reconstructed embryos in Sr6D+SrCB activation group was slightly higher than that of other groups,but they were not significantly different(P>0.05).3.Effect of different culture mediums on in vitro development of reconstructed embryo. The result was that reconstructed blastulas were only obtained in the mCZB^* culture medium.4.Effect of the different sucrose concentrations on in vitro enucleating time and in vitro development of reconstructed embryo.The result was that average micromanipulation time of each oocyte was descending following sucrose concentrations increasing.Enucleate rates of different sucrose concentrations in micromanipulation medium were all achieved into 100%. Fuse rate of reconstructed embryo in this micromanipulation medium without sucrose was slightly higher than that of other with sucrose(P>0.05).In vitro development of reconstructed embryo was not made decided whether sucrose or not,and reconstructed blastulas were obtained.But in vitro development rate of reconstructed embryo in micromanipulation medium with 4%,5%sucrose concentrations were significantly lower than that of groups (P>0.05).5.Effect of different electro-fusion parameters on in vitro development of reconstructed embryo.The result was that in vitro development of reconstructed embryo in this strength 200V/mm,pulse 60μs,repetition one of electro-fusion parameters was slightly higher than that of other groups(P<0.05).6.Effect of different temperature processing of donor cells on in vitro development of reconstructed embryo.The result was that fuse rate and in vitro development rate of the room temperature group were significantly higher than that of other groups(P<0.05).Reconstructed embryo in the low temperature group and high group developed to 2-4 cells at most.In vitro development of reconstructed embryo was not very well in this temperature change group.7.Effect of different electro-fusion temperature on in vitro development of reconstructed embryo.The result was that fuse rate in 25℃group was significantly higher than that of 37℃groups(P<0.05).8.In vitro development of different donor cells reconstructed embryo following nuclear transplantation.The result was that 141 oocytes were micro-manipulated in the first group,98 cumulus cell reconstructed embryo and 17.9%reconstructed blastulas were obtained;89 oocytes were micro-manipulated in the second group,65 fibroblast cell reconstructed embryo and 8.9%reconstructed blastulas were obtained.9.In vivo development of reconstructed embryos in recipient KM mouse.The result was that reconstructed embryos were simultaneously transplanted into in vivo of 5 pseudocyesis KM mouse.Nidations were only detected in this B or C groups.These above results indicate that:(1) When superovulative Hormone dosages were used With 5.0IU,in vivo maturing rate of KM mouse oocyte is the best.(2) The optimistic enucleate time is the best after HCG injection 10h.(3) In vitro development of reconstructed embryo is the best after HCG injection 11h and 12h.(4) The optimistic activating agent Sr6D+SrCB is suitable for in vitro development of reconstructed embryo.(5) mCZB^* culture medium is most beneficial to the in vitro development of reconstructed embryo.(6) In vitro development of reconstructed embryo was not made decided whether sucrose or not in micromanipulation medium,3%concentration sucrose is most beneficial to shorten in vitro oocyte enucleate time,manifest DNA nuclear zone in the cytoplasm and make enucleated operation become quick and saving time.(7) Strength 200V/mn,pulse 60μs,repetition one of electro-fusion parameters is most beneficial to the in vitro development of reconstructed embryo.(8) Room temperature treatment of donor cell is most beneficial to the in vitro development of reconstructed embryo.(9) 25℃fuse temperature is most beneficial to the fuseμ rate and in vitro development of reconstructed embryo.(10) In vitro development of donor cell reconstructed embryo is better than that of fibroblast reconstructed embryo.(11) Reconstructed embryo were transplanted into one oviduct pseudocyesis KM mouse. Pregnancy evidence are not seen after 19-20d.Nidations are only detected in this B or C groups.