西藏小型猪胚胎成纤维细胞的分离培养及性别鉴定

目的探索和建立西藏小型猪胚胎成纤维细胞体外分离培养及性别鉴定的技术方法。方法取35d的西藏小型猪胚胎分离胚胎成纤维细胞,进行体外原代培养及传代培养,观察细胞成纤维细胞的形态和生长状况。根据猪Y染色体上性别决定基因SRY设计引物进行性别鉴定,同时以β珠蛋白作为内参基因,建立PCR反应体系鉴别胚胎的性别。结果西藏小型猪胚胎成纤维体外分离后,呈贴壁生长,快速增殖。PCR性别鉴定结果表明雄性胚胎细胞可扩增出一特异性SRY基因条带,而雌性则没有。该法可快速鉴定胚胎的性别,可用于体细胞克隆动物早期性别鉴定。结论研究结果表明利用胶原酶消化法所获得的西藏小型猪胚胎成纤维细胞可在体外稳定培养并传代,利用PCR鉴定猪胎儿性别具有简单、快速、准确的特点,可应用于克隆猪研究中体细胞系的早期性别鉴定。

Isolation of Tibet Minipig Fetal Fibroblasts and Sexy Identification

Objective To explore and to establish the method of Tibet minipig fetal fibroblast isolation and in vitro culture. Methods Porcine fetal fibroblasts were isolated from 35d Tibet fetus of a pregnant gilt and were cultured or subcultured in vitro to observe morphology and cell growth. With the SRY gene of Y chromosome as targeting gene and βglobin gene as reference gene,PCR method was used for sexy identification. Results Tibet minipig fetal fibroblasts growed quickly and attached well in vitro after isolation from 35d fetus. The results of PCR method for sexy identification showed a specific band for male fetal fibroblasts but not for female. This method can be used for sexy identification before somatic nuclear transfer. Conclusions The isolated Tibet minipig fibroblasts digested with collagenase IV could grow and proliferate in vitro. The research result indicates that PCR of SRY gene is a simple,fast and accurate method to identify the sex of pig fetal firoblast cell lines,and can be used in routine study on transgenic cloning pigs.