聚乙二醇细胞融合技术在牛手工体细胞克隆中的应用初探

本研究分3个实验。实验1对比了不同浓度的离子霉素对牛去透明带卵母细胞孤雌激活的效果；实验2比较了手工半卵切割法去核与显微半卵切割法去核的效果；实验3对聚乙二醇（PEG）细胞融合技术在牛手工体细胞克隆的应用进行了初步探讨。结果表明，对于去透明带牛卵母细胞孤雌激活，离子霉素浓度为2．0μmol／L组的激活效果最好，其囊胚发育率（29．4％）极显著地高于浓度为5．0μmol／L的对照组和0．5μmol／L组（囊胚率分别为8．3％和9．4％，P〈0．01）；采用半卵切割法去核，手工切割的半卵存活率（85．6％）与显微操作（90．3％）差异不大，切割速率（约100枚卵／h）相仿。对双半卵与体细胞进行同步融合处理时，应用50％浓度的PEG融合率（39．3％）极显著好于45％、55％和60％的PEG（融合率分别为0％、16．2％和5．3％，P〈0．01）。

Preliminary study of cell fusion by PEG on handmade somatic cell cloning in cattle

Three experiments were carried out to assess the efficiency of cell fusion by polyethylene glycol （PEG） in the bovine zona-free manipulation cloning technique. The result showed that, when zona-free oocytes were activated by different concentrations of ionomycin, blastocyst development rate in activation by 2 μmol/L of ionomycin was siginificantly different to 5 μmol/L or 0.5 μmol/L （29.4% vs 8.3%, 29.4% vs 9.4%, respectively, P〈0.01）, while no significant difference in cutting efficiency （about 100 ova per hour） and survival rates Of oocytes after bisection by handmade or micromanipulation （85.6% vs 90. 3%, P〈0.05）. When two ennucleated cytoplasts and one granulosa cell were fused by PEG, the fusion rate （39.3%） in 50% PEG was significantly higher than those in 45%, 55% and 60% of PEG （0%, 16.2% and 5.3%, respectively, P〈0. 01）.