Study On Improving Efficiency Of Nuclear Transplantation In Bovine

The aim of this study was to improve the efficiency of cloning transgene animals. We evaluated the development of reconstructed embryos by three aspects: the blastocyst rate, the blastomere number and the apoptotic index of blastomere. The relationship among telomerase activity of donor cells, maturation of oocytes, activation methods and efficiency of reconstructed embryos were studied in this paper. The results of this paper were as following.1. A vector including hTERT gene and EGFP gene was transfered into the bovine ear fibroblast cells, the hTERT expression were detected by RT-PCR and Western blotting assay. The telomerase activity assay showed hTERT was compatible with bovine telomerase-related factors required for functional telomerase activity in these cells. A prolonged life span cell line, HBC3, was established with nomal karyotype and the cell line exhibited normal anchorage-dependent growth.2. The growth curve of HBC3 was same as the normal bovine fibroblast cells (BFC) and still responsible to growth factors such as EGF and insulin. The doubling index of HBC3 was 5.1 or 4.7 when added EGF or insulin respectively. Addition of serum was necessary for maintaining the growth of HBC3 and the apoptosis assay indicated that the apoptosis of HBC3 was significantly(p<0.01)lower then the 12th BFC(bovine ear fibroblast cell).3. Reconstructed embryos used HBC3 as the donor cells can develop to blastocyst, and there was no significantly difference in blastocyst rate between HBC3 and the control group. However, the blastomere number and apoptosis index of blastocyst was significantly lower than the control group(p<0.05).4. ABF11 and ABF12 cell lines were obtained when BFC was treated with DMBA and TPA, the morphology of ABF11 was fibroblast-like, and the ABF12 was triangle-like. Telomerase activity in these cell lines was tested by telomerase activity assay. And the growth curve of cells cultured with serum indicated that the ABF12 cell line had a longer logarithmic growth phase and had lost the contact inhibition. The doubling index of ABF12 and BFC cell lines with serum were 5.3 and 4.3 respectively. The doubling index of ABF12 and BFC cultured without serum was 5.0 and -1.2 respectively. Compared with the control group, the ABF12 cell line has lost the serum dependency. The ABF12 were cultured in soft sugar for 14d, and the cell formed 172 clones in soft sugar, which indicated that the ABF12 cell line had lost the contact inhibition.5. Then ABF12 cell line was used as donor cells to reconstruct bovine embryos and blastocyst was obtained, the cleavage rate and blastocyst rate was significantly lower than the BFC group. The flurescence staining indicated that the blastomere number of ABF12 and BFC was not significantly different. However, the apoptosis index of blastocyst was significantly(p<0.05) higher than the control, and the index of ABF12 and BFC was 0.124 and 0. 81 respectively.6. The maturation culture medium of oocytes was added with EGF, and the addition of EGF significantly improved the maturation rate of bovine oocytes and the subsequent development of reconstructed embryos. The subsequent blastocyst rate of oocytes cultured with EGF was significantly higher than the control. And the blastomere number of EGF group was higher than the control. Compared with the control, the apoptosis index of EGF group (0.062) was significantly lower. The oocytes cultured with EGF were cyropreserved, and the rate of normal morphology oocyte, the cleavage rate and blastocyst rate of the IVF embryos were significantly improved. The addition of VE did not significantly improve the maturation rate of bovine oocytes and the subsequent development of reconstructed embryos. And the VE in oocyte maturation had no significantly effect on the blastomere number. The normal morphology of cryopreserved oocytes, the cleavage rate and blastocyst rate of the IVF embryos were no difference between VE group and the control group. The addition of OCS in oocytes maturation medium had same effects as EGF.7. The effect of different concentration of bovine sperm extracts (BSE) on the reconstructed embryos were compared and the results indicated that BSE was able to active the SCNT embryos and supported the reconstructed embryos develop to blastocyst, the recommended concentration of BSE was 5mg/ml, the cleavage rate and blastcyst rate were significantly higher than the two others group. And the blastomere number of the 5mg/mL group was significantly(p<0.05) higher than the control, and the blastomere apoptosis index was 0.071, which was significantly lower than the control(p<0.05). The cleavage rate, the blastocyst rate, the blastomere number and the apoptosis index of the reconstructed embryos showed no significantly difference when BSE was combined with other activation methods.8. Effects of fusion activation methods and chemical activation methods were compared, and the cleavage rate and the blastocyst rate were significantly higher when the two activation methods were used together. The cleavage rate and blastocyst rate of injecting BSE were not significantly different with the chemical combined with fusion activation method. But the blastomere number of blastocyst activated by BSE was significantly higher(p<0.05) than the control, and the apoptotic index was significantly(p<0.05) lower than the control.9. 20ng/ml EGF was added into the SCNT embryos culture medium, the blastocyst rate of the reconstructed embryos was significantly higher(p<0.05)than the control. And the blastomere number of the control group and the EGF group were different significantly(p﹤0.05).The apoptotic index of the EGF group was 0.062, which was significantly lower than the control. The cryopreserved embryos survival rate after 24h and 48h in EGF group were significantly higher than the control. Addition of 0.1μg/ml and 1μg/ml progesterone in SCNT embryos culture medium significantly improved the blaststocys rate, there was no significantly difference with the control group in blastomere number and blastomere apoptotic index. Compared with the control group, addition of 100μg/ml VE in embryo culture medium showed no difference in SCNT embryo cleavage rate, but significantly(p<0.05)improved the blastocyst rate and the blastomere number. Moreover, the addition of VE significantly(p<0.01)reduced the apoptotic blastomere number and apoptotic index.10. 0.1 mol/L trelecose and fucose were added into the cryopreservation medium respectively, the survival rate after 24h and 48h of the cryopreserved SCNT embryos were significantly higher than the control. Different development stage of SCNT embryos were cryopreserved respectively, and the survival rate after 24h and 48h of morula were significantly lower than the blastocyst groups..