山羊胎儿成纤维细胞转染人t-PA指形区缺失基因及其核移植研究

采用阳离子脂质体法将人t-PA指形区缺失基因乳腺特异性表达载体(pEBT)导入山羊胎儿成纤维细胞,以山羊胎儿成纤维细胞和转染的山羊胎儿成纤维细胞作供体,构建核移植胚,对其体外发育情况进行了研究,比较了2种供体细胞(山羊胎儿成纤维细胞和转人t-PA指形区缺失基因的山羊胎儿成纤维细胞)及转人t-PA指形区缺失基因的山羊胎儿成纤维细胞饥饿处理与否对核移植胚胎体外发育的影响。结果表明,早期核移植胚有荧光蛋白(GFP)的表达;以山羊胎儿成纤维细胞作供体细胞时,核移植胚的桑葚胚率(50.3%)及囊胚率(16.0%)均高于以转人t-PA指形区缺失基因胎儿成纤维细胞为供体时的桑葚胚率(48.4%)和囊胚率(10.9%),但差异不显著(P>0.05);转人t-PA指形区缺失基因的山羊胎儿成纤维细胞经饥饿处理后,其核移植胚胎的卵裂率(73.6%)与不饥饿时的卵裂率(73.9%)差异不显著;饥饿处理后核移植胚胎的桑葚胚率(48.5%)和囊胚率(11.2%)均高于不饥饿处理的桑葚胚率(39.2%)和囊胚率(9.2%),但差异不显著(P>0.05)。本研究成功地构建了转人t-PA指形区缺失基因的体细胞核移植胚胎,体外囊胚率为11.2%。

Study of transfer t-PA gene to the goat fetal fibroblasts and nuclear transfer

Goat fetal fibroblasts were transfered into mammary gland expressed vector pEBT t-PA gene by using lipofectamin.The development of nuclear transfer embryos derived from fetal fibroblasts including t-PA gene were studied.And transgenic fetal fibroblasts were used to make nuclear transfer embryos;The development of nuclear transfer embryos derived from fetal fibroblasts including t-PA gene,fetal fibroblasts were compared.The development of nuclear transfer embryos derived from fetal fibroblasts including t-PA gene in medium DMEM including 0.5% FBS for starvation and including 10% FBS were compared.The result showed:nuclear transfer embryos were saw fluorescent protein(GFP);The morulae rate((50.3%)) and blastocyst rate (16.0%) of fetal fibroblasts cells were higher than that of fetal fibroblasts including t-PA gene(48.4%,10.9%)(P>0.05);Morular rate(48.5%) and blastocyst rate(11.2%) nuclear transfer embryos derived from fetal fibroblasts including t-PA gene cultured in medium DMEM including 0.5% FBS were higher than that(39.2%,9.2%) in media DMEM including 10% FBS(P>(0.05)).The transgenic nuclear transfer embryos were reconstructed,and blastocyst rate of transgenic nuclear transfer embryos was 11.2%.