转人t-PA指形区缺失基因的山羊体细胞核移植及其继代核移植

Somatic Nuclear Transplantation and Serial Nuclear Transplantation of Human Finger-domain Lacking t-PA Gene in Goat

摘要:

为了探索转基因体细胞核经连续核移植后的发育潜力，以转人组织型纤溶酶原激活剂（t-PA）指形区缺失基因的山羊胎儿成纤维细胞为核供体， MII期的卵母细胞质为核受体，利用胞质内注射法构建原代核移胚胎(G0)，并进行了原代核移植胚胎的继代核移植研究。比较原代和继代核移植胚胎在体外发育能力上的差异；在G1 、G2代核移植试验过程中，比较了供体胚胎细胞的发育阶段对核移植胚胎体外发育的影响。结果表明，原代核移植胚胎的卵裂率(76.45%±1.17%)与继代核移植胚胎的卵裂率(72.18%±1.97%，76.05%±2.38%，75.99%±2.84%)无显著性差异(P>0.05)。但原代核移植胚胎的桑葚胚率(47.20%±2.93%)、囊胚率(11.00%±1.42%)显著高于G₁、G₂、G₃代核核移植胚胎的桑葚胚率(34.99%±2.66%，28.23%±2.00%，23.34%±1.99%)、囊胚率(3.87%±0.67%，2.08%±1.66%，0)；在G₁、G₂中，当用16-细胞期核移植胚胎作为核供体时的桑葚胚率(29.57%±1.53%, 24.43%±1.87%)、囊胚率(1.96%±1.31%, 2.01%±1.34%)低于用32～64-细胞时期的核移植胚胎的桑葚胚率(34.32%±1.31%, 29.76%±1.66%)、囊胚率(3.86%±1.03%, 3.48%±0.34%)，但无显著性差异 (P>0.05)。由此得出结论：转基因体细胞核移植胚胎不宜进行多代克隆；胞质内注射法构建核移植胚胎，用32～64-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率高于用16-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率。

Abstract:

In order to research developmental competence of transgenic somatic cell by serial nuclear transplantation, goat cloned embryos were compared with recloned embryos in ability of in vitro development. Fetal fibroblasts including human finger-domain lacking t-PA gene was microinjected into cytoplasm of the MII oocytes. Goat embryos (G₀) were cloned by this procedure. A single blastomere from 16～64-cell goat cloned embryos(G₀) was microinjected into Intracytoplasm of the MII oocytes. Goat embryos (G₁) were cloned by this procedure. Goat embryos (G2, G3) were recloned by using 16～64-cell recloned embryos. The developmental time of donor embryo affected the developmental rate of recloned embryos(G₁,G₂).The results show: the cleavage rate of cloned embryos(G₀) (76.45%±1.17%)was no difference significantly with recloned embryos (G₁ G₂ G₃) (72.18%±1.97%，76.05%±2.38%，75.99%±2.84%); the developmental rate of morulae and blastocysts of cloned embryos (47.20%±2.93%,11.00%±1.42%) were higher than these of recloned embryos(34.99%±2.66%，28.23%±2.00%，23.34%±1.99%)(3.87%±0.67%，2.08%±1.66%，0); the morulae rate(29.57%±1.53%, 24.43%±1.87%) and blastocysts rate(1.96%±1.31%, 2.01%±1.34%) of recloned embryos (G₁ G₂) from 16-cell recloned embryos were lower than those(34.32%±1.31%, 29.76%±1.66% and 3.86%±1.03%, 3.48%±0.34%)from 32～64-cell recloned embryos(Ｐ>0.05). In conclusion, nuclear transfer embryos should not were recloned mostly; and the embryos recloned by using 32～64-cell embryos achieved higher developmental ability compared with using 16-cell embryos.