奶山羊胎儿成纤维细胞的分离培养及SRY基因性别鉴定

为了探索和建立奶山羊胎儿成纤维细胞体外分离培养及性别鉴定的技术方法,获得转基因克隆羊的供体细胞,本试验用组织块培养法分离纯化得到两株奶山羊胎儿成纤维细胞系,进行细胞形态观察、生长曲线及细胞周期和倍性分析。同时根据GenBank上发布的山羊SRY基因设计合成一对PCR引物作为性别鉴定引物,另外根据山羊BLG基因序列设计一对引物作为内参引物,建立PCR反应体系对两株胎儿成纤维细胞系进行性别鉴定。结果表明,分离的胎儿成纤维细胞活力良好,可在体外快速生长、增殖、稳定培养;阳性对照和山羊胎儿成纤维细胞系2经PCR扩增得到337 bp片段和498 bp的BLG基因片段,而阴性对照和山羊胎儿成纤维细胞系1经PCR扩增得到498 bp的β-乳球蛋白基因片段。将337 bp片段和pMD19-T载体连接,构建重组载体pSRY,通过测序证明337 bp片段为SRY基因片段。这说明有337 bp扩增带的细胞系为雄性,无337 bp扩增带的细胞系为雌性。本试验为转基因奶山羊新品种的培育奠定了基础。

Isolation and Culture of Diary Goat Fetal Fibroblasts and Sex Indentification by SRY Gene

In order to explore and establish the method for isolation,cultivation,and sex identification of dairy goat fetal fibroblasts(GFFs) in vitro,obtain the donor cells for dairy goat transgenic cloning,two dairy goat fetal fibroblast cell lines passing through separation and purify were obtained by tissue explant attachment.The GFFs were examined by cell morphology,growth curve,cell cycle and DNA ploidy.A pair of PCR primers were designed to identify the sex of GFF cell lines according to the goat SRY gene,the ...