绵羊体细胞核移植去核前程序的优化

Program optimization before enucleation on ovine somatic cell nuclear transfer

摘要:

目前绵羊体细胞克隆效率仍然很低，本研究拟对去核前的操作环节进行优化。主要为卵巢保存时间 (3 h和3–5 h)、卵母细胞体外成熟时间 (18 h和24 h)、供核细胞贴壁率 (10%和30%) 和盲吸法去核时间 (16 hpm和18 hpm) 等4个方面优化。以成熟率、融合率和重构胚胎发育能力作为评价参数。结果表明：在卵巢保存方面，卵巢保存3 h组卵母细胞成熟率显著高于3–5 h组卵母细胞成熟率 (60.18% vs 52.50%) (P<0.05)，重构胚胎发育力差异不显著 (P>0.05)；在体外成熟时间方面，体外成熟18 h组和24 h组卵母细胞成熟率差异极显著 (53.81% vs 89.06%) (P<0.01)，胚胎发育力差异不显著 (P>0.05)；在融合率方面，贴壁率30%组极显著高于贴壁率10%组 (80.85% vs 57.69%) (P<0.01)，在克隆胚胎发育率方面没有显著差异 (P>0.05)，具有贴比率差异性的细胞在细胞生长平台期表现出差异性；在去核时间方面，16 hpm组和18 hpm组胚胎卵裂率差异显著，囊胚发育力差异不显著 (P>0.05)，16 hpm组获得一只克隆羊，重复16 hpm获得4只妊娠克隆羊。组织微卫星序列经SDS-PAGE分析，DNA指纹与供体细胞相同。结论：去核前程序的优化保证了材料的质量，为提高克隆胚胎数量和质量奠定基础，可以获得体细胞克隆羊。

Abstract:

Ovine somatic cell nuclear transfer (SCNT) efficiency remains lower. Therefore, we optimized the program before oocyte enucleation on ovine SCNT. Four experiments were done including exposure duration of ovaries (3 h or 3 to 5 h), duration of oocytes maturation (18 h and 24 h), and rate of donor adherent and enucleation time of maturate oocyte. The maturation rates of oocyte, fusion rates and cleavation rates of cloned embryos were used to assess the efficiency of different procedures. The maturation rates of ovaries with 3 h exposure was higher than that of 3 to 5 h (60.18% vs 52.50%) (P<0.05). Embryonic development competence had no significant difference (P>0.05). The maturation rates were significantly different between group18 h and 24 h (53.81% vs 89.06%, P<0.01). Embryonic development competence had no significant difference (P>0.05); fusion rates of donor adherent 30% group was higher than that of 10% group. Embryonic development competence had no significant difference (P>0.05). Different adherent donor characterizes the difference in plateau phase. The cleavation rates of 18 hpm group was higher than that of 16 hpm group. Embryonic development competence had no significant difference (P>0.05), the enucleation of 16 hpm group obtained one clone fetus,we got four clone fetus to repeat the 16 hpm group. Five microsatellite was analyzed by PAGE,the bands indicated that fingerprint of cloned fetus were completely the same as those of donor cells. Our data therefore suggests program optimization before enucleation assurance quality of material which be able to improve the quantity and quality of clone embryos, and optimized scheme can obtain clone sheep offspring.