梅山猪胎儿成纤维细胞的分离培养及SRY-PCR法快速性别鉴定

对梅山猪胎儿成纤维细胞的分离培养及性别快速鉴定进行研究,为开展猪体细胞克隆、诱导多能干细胞获取及转基因等提供素材。通过取组织块培养法和胰蛋白酶消化培养法均成功得到梅山猪的胎儿(胎龄35 d)成纤维细胞,取其中7个梅山猪(msz4、msz5、msz6、msz9、msz11、msz14和msz15)胎儿的原代成纤维细胞,在传代培养至第5代时收集贴壁生长的细胞,提取基因组DNA。再利用根据SRY及ZFY/X设计的共2对引物进行PCR反应,扩增后进行琼脂糖凝胶电泳,出现166 bp和445/447 bp两条带的为雄性,只有445/447 bp一条带的为雌性。结果表明梅山猪的细胞系msz6、msz9和msz14为雌性细胞系,而msz4、msz5、msz11及msz15为雄性细胞系。试验结果证明了该方法可应用于鉴定不同培养方法获得猪成纤维细胞的性别,可为试验后期的转基因细胞制备提供性别依据。

Rapid sex identification by SRY-PCR of in vitro cultured Meishan pig fetal fibroblast

The present study was designed to obtain Meishan pig fetal fibroblast cells followed by identifying sex by SRY-PCR, in order to provide enough start materials for future somatic cell cloning, induced pluripotent stem cells and transgenesis in pigs. Meishan pig fetal (fetuses at gestational age of around 35 d) primary fibroblasts were obtained successfully by both explants seeding and trypsin-EDTA digestion seeding methods. To determinate the sex of seven cell lines from seven different Meishan pig fetuses, genomic DNA was separately extracted from passage five cells. Two pairs of primers were designed according to specific fragments in SRY and ZFY / X. After the PCR, two bands of 166 bp and 445/447 bp is for the male while single band of 445/447 bp is for a female in agarose gel electrophoresis. The results showed that msz6, msz9, msz14 were female cell line and msz4, msz5, msz11, msz15 were male cell line. The results indicated that the sex of pig fibroblasts from early fetuses could be successfully indentified, which will provide sexed donor cells for future studies in cloning, reprogramming and transgenesis.