应用ICSI技术生产转基因水牛胚胎的初步研究

本研究旨在探讨水牛精子完整质膜和破损质膜的方法对水牛单精子注射(ICSI)技术转基因效果的影响。水牛精子经冻融、Triton×-100、超声波3种方法破损精子质膜后与外源质粒DNA混合,采用ICSI的方法将基因转染后的精子注射到水牛体外成熟卵内,观察水牛ICSI卵激活后的发育状态和外源基因的表达效果。结果表明:精子质膜完整性实验中,冻融破损精子质膜组早期胚胎基因表达率为21.8%,显著高于活精子组的5.1%(P<0.01);冻融组与活精子组的卵裂率、囊胚发育率均无显著差异(P>0.05)。以冻融、Triton×-100、超声波3种方法破损精子质膜,冻融组的囊胚发育率(16.7%)最高,显著高于Triton×-100组(P<0.01)。同时,冻融组的早期胚胎基因表达率亦显著高于Triton×-100组和超声断尾组(60.3%vs.31.7%vs.18.2%,P<0.01)。上述结果说明,使用ICSI技术转外源基因可获得表达EGFP基因的水牛早期胚胎;冻融精子质膜破损法能有效破损精子质膜,有利于外源DNA与精子的结合,且提高转基因效率。

Primary Study on Production of Transgenic Buffalo Embryo by ICSI

Effects of sperm membrane and sperm membrane damaging methods on efficiency of transgenesis by ICSI in water buffalo were investigated.The results show that: When sperm membrane was disrupted by freeze-thawing method,the EGFP expressing rate was 21.8% significantly higher than that of live sperm 5.1%(P<0.01),while the cleavage rate,blastocyst development rate was no remarkable difference(P>0.05).When sperm membrane was disrupted by the freeze-thawing,Triton ×-100 and sonication respectively,the blastocyst development rate of freeze-thawing group was significantly higher than Triton ×-100 group's(16.7% vs.3.1%,P<0.01).And the EGFP expressing rate of freeze-thawing group was significantly higher than that of the other two groups(60.3% vs.31.7% vs.18.2%,P<0.05).These results indicate that,the EGFP expressing embryos of water buffalo could be generated by ICSI method;the efficiency of transgenesis by ICSI in buffalo can be improved by disrupted sperm membrane through freeze-thawing.