水牛体细胞核移植的研究

Studies on the Nuclear Transfer of Buffalo Somatic Cells

系统探讨了水牛体细胞核移植的各种影响因素 ,并初步建立了水牛体细胞核移植的一整套程序。体外成熟培养2 2～ 2 4 h的水牛卵母细胞去核后 ,将经血清饥饿 (0 .5 % FBS)培养 2～ 9d、0 .1mg/L Aphidicolin(APD)培养 +0 .5 %FBS培养 2～ 9d或一般培养法 (10 % FBS)培养的水牛耳皮成纤维细胞或颗粒细胞 ,直接注射到去核的卵母细胞质中 ,或注射到卵周隙中再经电融合 (10 0 V/mm ,15μs,电脉冲 3次 )构建重构胚。重构胚经化学激活后 (5μmol/L离子霉素 5 min,2 m mol/L 6 - DMAP3h)培养 7～ 8d,评定其胚胎发育能力。耳皮成纤维细胞和颗粒细胞经 0 .1mg/LAPD+0 .5 % FBS培养处理后的重组胚卵裂率 ,均极显著高于血清饥饿和一般培养处理的同种供体细胞 (P<0 .0 1) ,但囊胚发育率无显著差异 (P>0 .0 5 )。耳皮成纤维细胞和颗粒细胞经 0 .1mg/L APD+0 .5 % FBS处理后进行核移植的分裂率和发育率均无显著差异 (6 3.0 6 %比 5 8.70 % ,P>0 .0 5 )。以水牛颗粒细胞为核供体时 ,电融合法的重构胚分裂率显著高于胞质内注入法 (P<0 .0 5 ) ,囊胚发育率无显著差异 (P>0 .0 5 )。培养 3代和 6代的水牛颗粒细胞以及培养 6代和 10代的耳皮成纤维细胞 ,其具有正常二倍染色体的细胞比例均无显著差异 (P>0 .0 5 ) ;以这

This study was undertaken to systematically examine factors affecting the nuclear transfer of buffalo somatic cells,so as to establish a preliminary procedure for buffalo cloning.Buffalo oocytes obtained from ovaries at slaughter were matured in vitro for 22-24 h and then enucleated in manipulation medium containing 5 μg/mL cytochalasin B.Fibroblasts and cumulus cells after 3-9 passages in DMEM+10% FBS were treated by serum starvation(0.5% FBS for 2-9 days),0.1 μg/mL aphidicolin(APD) for 1 day and 0.5% FBS for 2-9 days or continue culturing in 10% FBS for 2-9 days,then,were transferred into enucleated oocytes by microinjection or electronic fusion(100 V/mm,15 μs and 3 pulses).The reconstructed embryos were activated by exposuring them to 5 μmol/L ionomycin for 5 min and then culturing in 2 mmol/L 6-DAMP for 3 h.After activation,embryos were cultured in 30 μL drop of granulosa cell monolayers for 8-10 days,to evaluate their cleavage and embryonic development.The cleavage rate of embryos reconstructed with fibroblasts and cumulus cells pretreated with 0.1 μg/mL APD+0.5% FBS were significantly higher than that of serum starvation group and control group(P<0.01),however there was no significant difference in the percentage of cleaved oocytes developing to blastocysts among the 3 groups.There was not significantly difference in the cleavage rate and embryonic development among embryos derived from fibroblasts and cumulus cells pretreated with 0.1 μg/mL APD+0.5% FBS(63.06% vs 58.70%,P>0.05).The cleavage rate of embryos reconstructed with cumulus cells by ellectrofusion was significantly higher than that by microinjection(P<0.05),but no difference was found in their proportion developing to blastocysts.Sixty-five percent to 85% of cumulus cells at 3 and 6 passages,and fibroblasts at 6 and 10 passages had normal karyotype,which did not show significant difference among them(P>0.05).When cumulus cells at G_3,G_4,G_5 and G_6 of passages were used as donor cells,the cleavage rate and blastocyst rate was similar,moreover,fibroblast cells at G_6,G_7,G_8 and G_9 of passages also resulted in a similar cleavage rate and blastocyst development.These results indicate that:(1)Fibroblasts and cumulus cells can be cultured up to 9 passages and keep relatively stable karyotepe;(2)Using 0.1 μg/mL APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system;(3)Both of fibroblast and cumulus cells can be used as the donor karyoplasts for nuclear transfer,and their efficiency are not influenced by the culture passages;(5)The development of reconstructed embryos by electrofusion is higher than that by microinjection,but there is no difference in the total efficiency between the two methods.