绵羊去透明带卵母细胞体细胞核移植研究

为探索简化的核移植程序,本研究分析不同成熟培养时间、去核过程中紫外光照时间、融合电压强度、激活剂种类、不同培养方法对绵羊去透明带卵母细胞核移植的影响。结果表明:卵母细胞成熟培养18～19 h后去除透明带,其极体排出和附着效果最佳。采用1.9 kV/cm直流电压融合去核卵母细胞与颗粒细胞,融合率为88.2%,效果最好。离子霉素对重构胚具有较好的激活效果,卵裂率为82.1%,囊胚率为10.4%;压制WOW(s孔中孔)发育组卵裂率和囊胚发育率与四孔板培养组相比无显著差异,但卵裂率和囊胚发育率并不高;去除透明带的绵羊卵母细胞采用衰减1/4的UV照射10 s辅助去核后,卵裂率为35.6%,但重构胚未能发育至囊胚。结果显示,通过去透明带辅助显微操作去核的方法进行的绵羊体细胞克隆程序较易掌握。

Somatic Cell Nuclear Transfer of Zona-free of Ovine Oocytes

Nuclear transfer were compared in this study in order to establish a simply nuclear transfer procedure,including oocytes maturation culture time,the time of oocytes exposed to UV,electric fusions,activated items and develpomental method.When oocytes were cultured for 18~19 h,the attaching rates of the first poly body and first poly body were appropriate for oocyte maturation.Electric fusions were performed in 1.9 kV/cm,the fusion rates were 88.2%.Enucleated zona-free oocytes and somatic cells were fused to reconstruct embryos,and the embryos were activated by Ionomycin,the cleavage and blastocyst rates were 82.1% and 10.4%.Reconstructed embryos were activated by the same methods after cultured in the WOWs(only pressed),there was no significant difference compared with that in four-well dish.When zona-free ovine oocytes was irradiated by UV(1/4) beyond 10s,most of ovine reconstruct embryos can not cleave to blastocyst after activation.The results indicated the zona-free cloning method might be easier to adopt than conventional cloning procedure for users,and was an easy procedure.