遗传 ›› 2013, Vol. 35 ›› Issue (6): 778-785.doi: 10.3724/SP.J.1005.2013.00778

• 研究报告 • 上一篇    下一篇

锌指核酸酶技术制备肌肉生长抑制素基因敲除的五指山小型猪成纤维细胞

曹随忠1, 岳成鹤1,2, 李西睿2, 冯冲2, 龙川2, 潘登科2   

  1. 1. 四川农业大学动物医学院, 雅安 625014 2. 中国农业科学院北京畜牧兽医研究所, 北京 100193
  • 收稿日期:2012-11-01 修回日期:2012-12-07 出版日期:2013-06-20 发布日期:2013-06-25
  • 通讯作者: 曹随忠 E-mail:pandengke2002@163.com
  • 基金资助:

    转基因动物新材料的育种价值评估项目(编号:2011ZX08010-006)和中央级公益性科研院所基本科研业务费专项(编号:2012yq-2)资助

Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases

CAO Sui-Zhong1, YUE Cheng-He1, 2, LI Xi-Rui2, FENG Chong2, LONG Chuan2, PAN Deng-Ke2   

  1. 1. College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China 2. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
  • Received:2012-11-01 Revised:2012-12-07 Online:2013-06-20 Published:2013-06-25

摘要: 敲除猪肌肉生长抑制素(Myostatin, MSTN)基因可能提高猪瘦肉率, MSTN基因敲除猪也可作为相关疾病的动物模型。文章利用锌指核酸酶(Zinc-finger nucleases, ZFNs)技术敲除五指山小型猪胎儿成纤维细胞MSTN基因, 为制备MSTN基因敲除猪奠定基础。ZFNs质粒或编码ZFNs的mRNA均能高效敲除MSTN基因, 使用ZFNs mRNA能直接得到MSTN+/-和MSTN-/-两种基因型的细胞克隆。DNA序列测定与分析发现, 细胞克隆的突变类型多为ZFNs作用靶位点处不大于10 bp的碱基插入或缺失(92.18 %); 氨基酸预测发现, 突变型MSTN基因的终止密码子常常提前出现。将MSTN基因敲除的细胞进行体细胞核移植(Somatic cell nuclear transfer, SCNT)发现, 胚胎体外早期发育潜力与野生型无显著差异, 表明这些细胞可用于后续MSTN基因敲除猪的制备。

关键词: 肌肉生长抑制素, 基因敲除, 五指山小型猪, 锌指核酸酶, MSTN-/-成纤维细胞

Abstract: Disruption of myostatin (MSTN) gene in pigs may improve porcine lean meat percentage (LMP), and create an animal model for certain human diseases. Using zinc-finger nucleases (ZFNs) technology, MSTN gene was deleted in Wuzhishan miniature pig fibroblasts by transfection of either ZFNs plasmids or ZFNs mRNA in high efficiency. Strikingly, ZFNs encoding mRNA could produce MSTN+/-and MSTN-/- cell colonies with single or double allele deletion by single transfection. Sequencing results demonstrated that 92.18% of the mutations were short fragment deletions or insertions (≤10 bp). Prediction of amino acids sequences indicated that more than half of the mutations cause premature transla-tional-termination codon. MSTN+/+, MSTN+/-, and MSTN-/- cell colonies were used as nuclear donor for somatic cell nuclear transfer (SCNT), and developmental potential of SCNT embryos were measured by the blastocyst rate. The results revealed no significant difference in development competence among the three kinds of reconstructed embryos (14.29% vs. 19.64% vs. 16.13%), which provides the possibility of making myostatin knock out pigs in the future.

Key words: zinc-finger nucleases (ZFNs), myostatin, gene knockout, Wuzhishan miniature pigs (WZSP), MSTN-/- fibroblasts