| This study were designed to investigate development potency of the reconstructed embryos of rat-mouse and rat-bovine inter-species nuclear transfer in vitro/vivo, to develop the principle and technology of somatic nuclear transplantation in inter-species of mammalian and to study the mechanism of coordination between cell nuclear and cytoplasm. Somatic nuclear transplantation technology has been used to establish stem cell from different animals, which can provide material to treat various diseases. The main contents and results were as follows:1. The establishment of SD rat fibroblast for the donor of nuclear transfer The method of single cell cloning by micro-manipulating was improved topurify fibroblast. As a result, steady fibroblast can be obtained and the rate of the clone formation is 92.71%.2. Synchronization treatment of SD rat fibroblast and karyotype analysis With serum starvation treatment, cells at Go+Gi phase occupy 74.5 ?4.4%. Whereas cells which were treated with nocodazole at G2/M were 45. 9 + 5. 21%. The results are notable. The results of Giemsa staining show the number and the shape of chromatosome was normal (2n=42).3. Somatic nuclear transfer of rat-mouse inter-species1) The purpose of this experiment was to study difference of development potency of embryos recombined by cytoplasm injection and electrical fusion. As a result: the rates to develop morula, which the reconstructed embryos were activated by 6-DMAP + CCB + ethanol, were 9.84% and 9.48%. This indicated that there was not a significant difference (P>0.05) for two methods to recombine rat-mouse inter-species somatic cell nuclear transfer embryos.2) The rat-mouse reconstructed embryos activated were been cultured in the maturation mediums of TCM199, Ml6, CZB and CZBM(CZB modified) for 48h. The cleavage rate were 20.83%, 16.93%, 22.67%. 35.16% and the development rate to morula were 6.09%, 2.11%, 10.55%,10.93%, respectively. Results of the experiment show that CZBT were much suitable than the others.3) The donor of SD rat fibroblast cells, which had been treated with serum starvation for 3-5d ,the 10 u g/ml of nocodazole and control, were used to recombine rat-mouse nuclear transfer embryos. After the recombined embryos were activated by 6-DMAP + CCB + ethanol, the rates of morula were 4.14%, 6.45% and 2.94%, respectively.4) The enucleated rate (88.23%) of the mouse maturation oocytes were improved in the medium containing 10?ug/ml CCB. 6-DMAP+CCB+ethanol can effectively activate the reconstructed rat-mouse embryos.5) 225 reconstructed embryos from 2-cell to morula were transferred into 46 recipients. No Pregnant rats had been observed for 20d. This event indicated mouse enucleated maturation oocytes can't support the donors of rat fibroblast cells to develop a viable offspring.4. Rat-Bovine interspecies somatic nuclear transfer1) Bovine COCs had been cultured in the maturation medium containing 8-10% OCS in vitro for 22-24h, 60.86%- 61.41% of maturation oocytes can be obtained.2) we got 55.08% of fusion rate when reconstructed embryos were fused with 1500-1800V/cm voltage and 50ms impulse. In addition, a same result can be obtained with low voltage +length impulse or strong voltage +short impulse.3) Recombined embryos were activated by hyalidase, cycloheximide and 6-DMAP+ethanol+CCB.The cleavage rates were 3.70%. 15.27%, 18.21%, the morula rates were 0.00%, 9.25%, 8.92%, respectively. This data showed that 6-DMAP+ethano+cycloheximide has stranger effect on reconstructed embryos of inter-species than others'.4) After the activated nuclear transfer embryos had been cultured in M199 ^ and CZBM for 48h, the cleavage rate can reach at 16.41% and 17.41%, and the morula rate were 7.46%,% and 6.74%, respectively.It has been reported that the viable offspring has been cloned successfully mammalian somatic nuclear transfer of cross-species. In this study, we found that enucleated maturation oocytes of bovine and mouse can only support nuclear transfer embryos to develo... |
PDF Full Text Request