不同融合条件及激活方法对体细胞克隆绵羊胚胎发育的影响

Effect of Different Fusion Conditions and Activated Methods on the Embryo Growth of Sheep Cloned from Somatic Cells

对成年绵羊耳部皮肤细胞进行培养传代 .将培养出的成纤维细胞经血清饥饿法处理后 ,作为核供体移植到MII期去核卵母细胞中进行核移植胚胎的生产 .用细胞松弛素B法与离子霉素 + 6 DMAP法对核移植胚胎 (重构胚 )在电融合后进行化学激活 ,然后在 38.6℃、5%CO2 、最大相对湿度条件下 ,进行体外培养 (IVC) .重构胚卵裂率分别为 37.8%与 43.8% ( 51 /1 35与 81 /1 85) ,囊 /桑葚胚率分别为 9.8%和 1 2 .3% ( 5/51和 1 0 /81 ) .在融合电压 1 .5kV/cm、持续时间 30μs、脉冲次数为 2次的条件下 ,孤雌激活胚胎的卵裂率和囊 /桑葚胚率分别为 81 .0 %和1 2 .9% .

Adult sheep ear skin cells were cultured and passaged. Fibroblast cells were transferred to enucleated oocytes by micromanipulation and electrofusion. The donor cells used for nuclear transfer were synchronized in presumptive G 0 by serum starvation. Cytochalasin B and ionomycin plus 6 dimethylaminopurine (6 DMAP) were used as activation agent. Nuclear transplantation units were cultured in 38.6℃, 5%CO 2,95% air, humidified incubator. Reconstructed embryos have a cleavage rate of 37.8% and 43.8%(51/135 and 81/185),morulas/blastocysts rate of 9.8% and 12.3%(5/51 and 10/81)respectively. Parthenogenetically activated oocytes have a 81.0% and 12.9% of cleavage rate and morulas/blastocysts rate on the condition of two pulses direct current of 1.5 kV/cm for 30 μsec each by an electro cell manipulator.