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

生物技术通报 ›› 2013, Vol. 0 ›› Issue (5): 116-120.

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

信吉阁^{1, 2}, 肖晶², 查星琴^{1, 2}, 成文敏^{1, 2}, 卿玉波^{1, 2}, 潘伟荣^{1, 2}, 曾养志², 魏红江^{1, 2}

(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)

收稿日期:2012-10-16 修回日期:2013-05-24 出版日期:2013-05-24 发布日期:2013-05-24
作者简介:信吉阁，女，博士研究生，实验师，研究方向：胚胎生物技术；E-mail：synlelovely@yahoo.com.cn
基金资助:
云南农业大学动物科学技术学院科研基金项目(SW000126)

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

Xin Jige^{1, 2}, Xiao Jing², Zha Xingqin^{1, 2}, Cheng Wenmin^{1, 2}, Qing Yubo^{1, 2}, Pan Weirong^{1, 2}, Zeng Yangzhi², Wei Hongjiang^{1, 2}

(1. Animal Science and Technology College，Yunnan Agricultural University，Kunming 650201；2. Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province，Kunming 650201)

Received:2012-10-16 Revised:2013-05-24 Published:2013-05-24 Online:2013-05-24

摘要/Abstract

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。

关键词: 版纳微型猪近交系, 胎儿成纤维细胞, SRY基因

Abstract: 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.

Key words: Banna mini-pig inbred line, Fetal fibroblast, SRY gene

信吉阁, 肖晶, 查星琴, 成文敏, 卿玉波, 潘伟荣, 曾养志, 魏红江. 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[J]. 生物技术通报, 2013, 0(5): 116-120.

Xin Jige, Xiao Jing, Zha Xingqin, Cheng Wenmin, Qing Yubo, Pan Weirong, Zeng Yangzhi, Wei Hongjiang. Porcine Fetal Fibroblast Culture and Sex Identification of Banna Mini-pig Inbred Line[J]. Biotechnology Bulletin, 2013, 0(5): 116-120.

参考文献

[1] Wang L, Lu XF, Lu YR, et al. Immunogenicity and immune modulation of osteogenic differentiated mesenchymal stem cells from Banna minipig inbred line [J]. Transplant Proc, 2006, 38(7)：2267-2269.
[2] Yu P, Zhang L, Li SF, et al. Screening and analysis of porcine endogenous retrovirus in Chinese Banna minipig inbred line [J]. Transplant Proc, 2004, 36：2485-2487.
[3] Tan WD, Chen YN, Lu YR, et al. Expression sequence tags research of a cDNA library from liver tissue of Banna minipig inbred line [J]. Am J Transpl, 2006, 82(1)：1054-1055.
[4] 信吉阁.版纳微型猪近交系体细胞核移植的基础研究[D].昆明：云南农业大学, 2007.
[5] 卿玉波.不同品种受体母猪对版纳微型猪近交系核移植效果影响[D].昆明：云南农业大学, 2009.
[6] Betthauser J, Forsberg E, Augenstein M, et al. Production of cloned pigs from in vitro systems [J]. Nature Biotechnology, 2000, 18：1055-1059.
[7] Heyman Y, Chavatte-Palmer P, Lebourhis D, et al. Frequency and occurrence of late-gestation losses from cattle cloned embryos [J]. Biol Reprod, 2002, 66(1)：6-13.
[8] Yin XJ, Lee HS, Lee YH, et al. Cats cloned from fetal and adult somatic cells by nuclear transfer [J]. Reproduction, 2005, 129(2)：245-249.
[9] Kunieda T, Xian M, Kobayashi E, et al. Sexing of mouse preimplanta-tion embryos by detection of Y chromosome-specific sequences using polymerase chain reaction [J]. Biology of Reproduction, 1992, 46(4)：692-697.
[10] 司徒镇强, 吴军正.细胞培养[M].北京：世界图书出版公司, 2007.
[11] 尹智, 孔庆然, 格日乐其木格, 等.应用双重PCR技术鉴定猪胎儿性别及其细胞系建立[J].东北农业大学学报, 2008, 39(9)：49-52.
[12] 张晟, 肖高芳, 黄黎珍, 等.借助慢病毒将EGFP基因导入西藏小型猪胎儿成纤维细胞[J].中国比较医学杂志, 2010, 20(4)：34-36.
[13] 李小平, 袁婷, 高飞, 等.双温多重PCR鉴定猪早期胚胎性别的研究[J].中国农学通报, 2010, 26(14)：34-36.
[14] 曹海峰, 随刘才, 季索菲, 等.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[J].安徽农业大学学报, 2011, 38(6)：907-910.