| Somatic cell nuclear transfer (SCNT) is a key technique for producing transgenic mammalian, so optimization of this technique is important for improving the total efficiency of transgenic animal production. In order to improve the efficiency of SCNT, in this study we optimized SCNT technique in several procedures, including oocyte in vitro mature, in vitro culture of cloned embryos, enucleation, treatment and selection of donor cells. As well, hLYZ and hBD3 transgenic bovine fibroblast cells were used as donor cells for production of transgenic cloned embryos and animals. In results, 20 recipients which maintained pregnancy for 2~8 months have achieved. Following results have been obtained:1. Three new born skin fibroblast cells and one fetal fibroblast cells at 40 d of gestation were isolated by attaching tissue explants. There was no difference in grow speed and kyaroytype among fibroblasts derived from three new born bovines. However, grow speed of fetal fibroblasts in primary culture was faster than new born skin fibroblast cells. After several passages, no different in grow speed was observed between new born skin fibroblast cells and fetal fibroblast cells. After SCNT, no significant difference was observed among the four fibroblast cells.2. The effect of bovine ovary storage temperatures: 15℃,20℃,25℃,30℃, during transport from abattoir to laboratory on the the quality of collected oocytes in term of the number of grade A and B oocytes, in vitro maturation and developmental competence after somatic cell nuclear transfer was compared. the percentage of grade A and B oocytes were 60.2% and 64.0% in 20℃and 25℃respectively, significantly higher than these collected from ovaries storage in 15℃and 30℃(48.4%,40.1%, P<0.05). After SCNT, no significant difference in Cleavage (76.0%,75.0%,74.5%) and blastocyst rates (29.2%,20.2%,22.4%) when oocytes collected from ovaries storage in 15℃,20℃,25℃were used as recipient cytplasts, significantly higher than these from 30℃group (cleavage and blastocyst rate were 43.8% and 8.3% respectively) (P<0.05).3. Supplying 10 ng/mL leptin in vitro oocyte mature medium can significantly improve blastocyst development rate of cloned embryo (30.4% Vs 17.3%, P<0.05).4. The effects of two culture media: mSOF and G1.1/G1.2, on the development of somatic cell cloned embryos were compared. In results, there were differences in developmental speed and shape when embryos were cultured in different media. Dvelopmental speed of cloned embryos was faster in G1.1/G1.2 The levels of compaction in morula and expand in blastocyst were more obvious in mSOF medium. Although no significant difference was observed in cleavage and blastocyst development rate between these two culture media, hatching rate was significantly higher in mSOF medium(31.5% vs 21.3%, P<0.05); apoptotic index was significantly lower in blastocysts derived from G1.1/G1.2 media(8.5±1.2% vs 16.8±1.5%, P<0.05);There was no effect of culture media on the ratio of ICM and TE in blastocysts. Although no significant difference in pregnancy rate was observed at 40 d of gestation, the pregnancy rate was significantly higher 60 d after embryo transfer in G1.1/G1.2 media group(23.3% vs 46.1%,P<0.05).5. After treatment with demecolcine, most treated oocyte (89.3%) can observed cone aroud nuclear area. The enucleation rate was 98.5% in demeclocine-assisted enucleation method group, significantly higher than blind enucleation method(P<0.05).6. When cells with diameter in 15～20μm were selected and used as nuclear transfer donor cells, the blastocyst rate was significantly higher compared with donor cells with 10μm or 25μm in diameter(P<0.05).7. Compared with traditional chamber fusion protocol, fusion rate in microelectrode fusion protocol was significantly higher (85.6% vs 66.7%, P<0.05).8. There was no significant difference in developmental competence of somatic cell cloned embryos when donor cells were treated by serum starvation, contact restrain, or no-treatment (P>0.05).9. We transfect mammary-specific expression vectors hLYZ and hBD3 that have been constructed in our laboratory separately into cow fibroblasts. Transgenic positive cells have been obtained by G418 selection for 4 weeks.10. No significant difference was found in fusion and cleavage rates between transgene transfected and non-transfected group. But the blastocyst development rate is lower in transfected group. (P<0.05). It indicated that transgene transfection has negative effect on development of transgenic cloned embryos.12. The expression of GFP was observed until the 4-cell stage of transgenic cloned embryos with a percentage of 7.8% (4/51), 40.9% (18/44) embryos could be observed GFP under UV in blastcytes stage.13. The effects of inherited background of the donor cells on the developmental competence of transgenic cloned embryos were compared. Results showed that there was no notable difference in blastocyst rates of transgenic cloned embryos between AFCs from two cattles; but the blastocyst rates of transgenic cloned embryos from FFC was significant higher than that from AFC (18.4%, 19.4% VS 34.0%, P<0.05); Moreover, pregnancy rates on day 60 were no significant difference regardless of the inherited background of the donor cells and AFC or FFC(7.69% VS 8.96%, P>0.05 ).14. The number of transfer with the hLYZ-Integrated and hBD3-integrated cloned embryos was up to 133 and 152 respectively, following 11 cf 133 pregnancy 2 months later and 9 of 152 pregnancy 3 months respectively...
|
PDF Full Text Request