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

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

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)。

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）.