版纳微型猪近交系胎儿成纤维细胞的分离培养及性别鉴定  

Porcine Fetal Fibroblast Culture and Sex Identification of Banna Mini-pig Inbred Line

作　　者：信吉阁[1,2] 肖晶 查星琴[1,2] 成文敏[1,2] 卿玉波[1,2] 潘伟荣[1,2] 曾养志 魏红江[1,2] 

机构地区：[1]云南农业大学动物科学技术学院,昆明650201 [2]云南省版纳微型猪近交系重点实验室,昆明650201

出　　处：《生物技术通报》2013年第5期116-119,共4页Biotechnology Bulletin

基　　金：云南农业大学动物科学技术学院科研基金项目(SW000126)

摘　　要：旨在建立版纳微型猪近交系猪胎儿成纤维细胞分离培养及性别鉴定的技术方法,采用胶原酶消化法分离版纳微型猪近交系猪胎儿成纤维细胞,同时根据GenBank上猪的SRY基因序列设计合成一对引物,并以β-actin基因为内参基因,利用多重PCR进行胚胎性别鉴定,并优化了PCR反应条件。结果表明,经胶原酶消化能成功分离版纳微型猪近交系猪胎儿成纤维细胞,2号、3号胚胎提取的DNA经多重PCR能扩出309 bp的SRY基因片段和132 bp的内参基因片段,为雄性胚胎,1、4、5、6、7号只扩增出内参基因,为雌性胚胎。PCR产物经测序与NCBI上发表的SRY基因序列比对,同源性为99%,进一步说明了性别鉴定结果的正确性。为转基因、基因敲除试验特定性别供体细胞的选择提供可靠的技术依据。The present study was designed to culture fetal fibroblast of Banna mini-pig inbred line and built sex identification method.Experiments were conducted to isolate fetal fibroblast of Banna mini-pig inbred line from embryos by collagenase digestion and identify their sex using multiplex PCR through optimizing conditions,in which two pairs of primer were designed relatively according to specific fragments in SRY gene and β-actin gene（as internal control）.The results demonstrated that it could successfully isolate fetal fibroblast of Banna mini-pig inbred line and the embryo of number 2,3 were male that could amplify specific DNA sequences of SRY gene（309 bp）and reference gene（132 bp） by multiplex PCR,and the number 1,4,5,6,7 were female with only reference gene.The PCR products were sequenced and aligned with SRY gene sequence published in NCBI.It shared 99% homology and this made to further verify the validity of results.The study provided a reliable technical basis for the culture of special sex fetal fibroblast in transgene and gene knock-out research.

关 键 词：版纳微型猪近交系 胎儿成纤维细胞 SRY基因 

分 类 号：S828[农业科学—畜牧学]