敲除山羊胎儿成纤维细胞中的抗体重链基因

Knockout of The Gene Encoding Immunoglobulin-mu in Goat Fetal Fibroblasts

在针对大动物的精确基因修饰研究中,基于体细胞的同源重组是唯一可行与有效的方法.其中,沉默基因位点的重组尤为困难.为获得抗体基因功能缺失的山羊用于人源化抗体的研究,通过体细胞同源重组技术,首次成功地获得了抗体基因敲除的山羊胎儿成纤维细胞株,该细胞株可用于体细胞克隆制备抗体基因功能缺失的转基因山羊.以35日龄的山羊胎儿成纤维细胞(GEF88)基因组DNA为模板,扩增山羊抗体重链J-Cμ基因作为同源臂,构建了同基因型的正负筛选打靶载体GTIgH.将此打靶载体经电穿孔的方法转染GEF88细胞,并通过0.8mg/L的嘌呤霉素进行药物筛选,获得了362个抗性细胞克隆,PCR、测序及DNA印迹鉴定结果显示,其中的GT211抗性细胞克隆为中靶细胞,该细胞克隆中的抗体重链基因的一条等位基因已被成功敲除.

Gene targeting in livestock fibroblasts has proven extremely difficult to achieve, particularly on silent gene locus. To obtain IgH functional disruption goats used for humanized antibody research, the goat IgH gene, which is transcriptionally silent in fibroblast, was knocked out and the targeted fibroblasts can be used to produce goats with IgH disruption through somatic cell clone. The goat immunoglobulin heavy chain J-Cμ fragment was amplified from the genomic DNA of goat fetal fibroblasts GEF88 through PCR and used as homologous arm to construct an isogenic Positive-Negative Selective targeting vector GTIgH. Then the GEF88 were transfected with the linearized targeting vector GTIgH through electroporation and selected in cell culture medium with 0.8 mg/L puromycin. 1 of the 362 drug-resistant clones was positive for targeting events through PCR screen, and the targeted clone was further confirmed by sequencing and Southern blotting. This suggests that one allele of IgH gene has been successfully knocked out in goat fetal fibroblasts.