应用双重PCR技术鉴定猪胎儿性别及其细胞系建立

Sex identification of porcine fetus by duplex-PCR method

sry和zfy/x是与哺乳动物性别决定有关的重要基因。本试验针对猪sry基因设计了两对PCR引物,以zfy/x为内参基因,利用双重PCR技术对6只30d左右猪胎儿的性别进行鉴定。结果表明,所有的6个样品都有447bp(雄性)或445bp(雌性)的zfy或zfx序列的扩增带,而采用两对不同的引物对sry序列扩增后,有1个样品出现241bp和166bp的扩增带为雄性,其余的无特异性扩增带为雌性,然后采用胶原酶消化培养法建立已知性别的猪胎儿成纤维细胞系。该方法具有简单、快速、准确的特点,为转基因克隆动物研究中尽早转染目的基因以及培养特定性别的胎儿成纤维细胞系提供了可靠的依据。

In mammals, sry and zfy/x are important to sex determination. Two pairs of PCR primers were designed and the amplification product of the zfy/x genes was used as a positive control for PCR to identify the sex of porcine fetuses by duplex-PCR method. The results showed that zfy/x genes as positive controls had the amplified 447/445 bp band in all samples, 1 of 6 samples amplified 241 bp and 166 bp hands were males and the others that didn＇t have amplified sequences were females, then porcine fetal fibroblasts were cultured by fetal tissue pieces which were digested with eollagenase. It indicates that the method is simple, fast and accurate, and can be used in routine study on transgenie cloning animals to define the sex of fetal fibroblasts early.