人血清白蛋白基因的打靶研究

为获得整合有人血清白蛋白(HSA)基因的猪胎儿成纤维细胞克隆,从猪基因组文库中杂交筛选得到猪血清白蛋白(PSA)基因全序列35kb并克隆了人血清白蛋白cDNA序列,扩增猪血清白蛋白基因5′调控序列7.2kb片段及第一内含子至第四内含子2.8kb的片段;构建了含有neo及tk正负筛选标记基因的人血清白蛋白基因打靶载体,并验证了neo基因的有效性。将线性化的打靶载体通过电击转染的方法整合到猪胎儿成纤维细胞基因组中,利用G418及GANC进行细胞克隆的抗药性筛选,PCR及Southern blot鉴定抗药性细胞克隆,最终获得3个发生同源重组的细胞克隆。这为下一步进行体细胞核移植制备生产人血清白蛋白转基因猪奠定了基础。

The Targeting Study of Human Serum Albumin Gene

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.