【摘要】 克隆了人lactoferrin基因和山羊β-casein基因5′端调控区,构建了人lactoferrin的乳腺表达载体,并将该载体利用脂质体介导转染了奶山羊胎儿成纤维细胞,获得了稳定整合人lactoferrin基因的转基因体细胞克隆17个,其中PCR和SouthernBlot检测阳性的细胞克隆14个,阳性率82.4%。以转基因体细胞为供体细胞进行了核移植,获得了能够体外发育的山羊转基因克隆胚胎,体内成熟卵母细胞来源的核移植囊胚率为64.8%,体外成熟卵母细胞来源的核移植囊胚率为51.7%,证明了山羊转基因体细胞能够支持克隆胚的进一步发育。更多还原

【Abstract】 Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat β-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.更多还原