Akshey Y S, Malakar D, De A K, Jena M K, Sahu S, Dutta R
Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
Reprod Domest Anim. 2011 Aug;46(4):699-704. doi: 10.1111/j.1439-0531.2010.01732.x. Epub 2010 Dec 6.
The present investigation was carried out to find an efficient chemically assisted procedure for enucleation of goat oocytes related to handmade cloning (HMC) technique. After 22-h in vitro maturation, oocytes were incubated with 0.5 μg/ml demecolcine for 2 h. Cumulus cells were removed by pipetting and vortexing in 0.5 mg/ml hyaluronidase, and zona pellucida were digested with pronase. Oocytes with extrusion cones were subjected to oriented bisection. One-third of the cytoplasm with the extrusion cone was removed with a micro blade. The remaining cytoplasts were used as recipients in HMC. Goat foetal fibroblasts were used as nuclear donors. The overall efficiency measured as the number of cytoplasts obtained per total number of oocytes used was significantly (p < 0.05) higher in chemically assisted handmade enucleation (CAHE) than oriented handmade enucleation without demecolcine (OHE) (80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). The reconstructed and activated embryos were cultured in embryo development medium (EDM) for 7 days. Fusion, cleavage and blastocyst development rate were 71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33% (mean ± SEM), respectively which did not differ significantly from those achieved with random handmade enucleation and OHE. In conclusion, chemically assisted enucleation is a highly efficient and reliable enucleation method for goat HMC which eliminates the need of expensive equipment (inverted fluorescence microscope) and potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection.
本研究旨在寻找一种高效的化学辅助方法，用于与手工克隆（HMC）技术相关的山羊卵母细胞去核。体外成熟22小时后，将卵母细胞与0.5μg/ml秋水仙胺孵育2小时。通过在0.5mg/ml透明质酸酶中吹打和涡旋去除卵丘细胞，并用链霉蛋白酶消化透明带。对具有挤出锥的卵母细胞进行定向二分。用微型刀片去除三分之一带有挤出锥的细胞质。剩余的细胞质体用作HMC中的受体。山羊胎儿成纤维细胞用作核供体。化学辅助手工去核（CAHE）的总体效率（以每使用的卵母细胞总数获得的细胞质体数量衡量）显著（p<0.05）高于未使用秋水仙胺的定向手工去核（OHE）（分别为80.02±1.292%和72.9±1.00%，平均值±标准误）。将重构并激活的胚胎在胚胎发育培养基（EDM）中培养7天。融合、分裂和囊胚发育率分别为71.63±1.95%、92.94±0.91%和23.78±3.33%（平均值±标准误），与随机手工去核和OHE所获得的结果无显著差异。总之，化学辅助去核是一种用于山羊HMC的高效且可靠的去核方法，它无需昂贵的设备（倒置荧光显微镜），也无需潜在有害的染色质染色和紫外线（UV）照射来选择细胞质体。
