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.PDFVIP

安徽农业大学学报, 2011, 38(6): 907-910 Journal of Anhui Agricultural University [DOI]CNKI：34-1162/S1025.004 网络出版时间：2011-10-25 10:25:14 [URL]/kcms/detail/34.1162.S1025.004.html 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 * 曹海峰，随刘才，季索菲，李运生，陶 勇，张运海 ，章孝荣 (安徽农业大学动物科技学院，动物胚胎工程实验室，合肥 230036) 摘 要：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。 关键词：成纤维细胞系；SRY；性别鉴定 中图分类号：S828.81 文献标识码：A 文章编号：1672−352X (2011)06−0907−04 Rapid sex identification by SRY-PCR of in vitro cultured Meishan pig fetal fibroblast CAO Hai-feng, SUI Liu-cai, JI Suo-fei, LI Yun-sheng, TAO Yong, ZHANG Yun-hai, ZHANG Xiao-rong (Laboratory of Embryo Engineer of Animal, School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036) Abstract: The present study was designed to obtain Meishan pig fetal fibroblast cells followed by identi- fying sex by SRY-PCR, in order to provide enough start materials for future somatic cell cloning, induced pluri- potent 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 fragme