CD163双等位基因编辑猪的制备及传代

魏迎辉1,2 刘志国1 徐奎1 Evanna Huyhn3 Paul Dyce3 李继良4 周伟良4 董树仁4 冯保亮4 牟玉莲1 Ju LangLi2,3 李奎1

(1.中国农业科学院北京畜牧兽医研究所, 中国北京 100193)
(2.佛山科学技术学院, 中国广东佛山 528000)
(3.Department of Animal Bio Sciences, University of Guelph, Canada Ontario N1G 2W1, Canada)
(4.天津市宁河原种猪场, 中国天津 301504)

【摘要】【目的】猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS),俗称“蓝耳病”,是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种高致死性传染病,对世界养猪业造成了巨大的经济损失。由于PRRSV遗传变异性较大,因此国内外并未有理想疫苗能够对此病进行有效防制。Cluster of differentiation 163(CD163)是PRRSV感染猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)的受体之一,研究旨在利用CRISPR/Cas9技术结合体细胞核移植技术制备CD163基因编辑的大白猪。【方法】针对猪CD163基因的第7外显子设计构建CRISPR/Cas9基因编辑载体;转染大白猪胎儿成纤维细胞,获得基因编辑阳性细胞克隆;以基因编辑细胞为核供体、体外成熟的猪卵母细胞为核受体构建克隆胚胎;胚胎移植到受体母猪生产CD163基因编辑猪,并进行后续的扩繁试验。【结果】设计的g RNA能够高效的识别靶位点。对获得的127个细胞单克隆进行PCR和测序显示,共有21个克隆发生突变,其中14个克隆为单等位基因突变或双等位基因杂合突变,7个克隆为双等位基因纯合突变。通过体细胞核移植技术,成功获得了CD163双等位基因编辑的纯合大白猪;并获得首批F1代CD163基因编辑仔猪,目前健康状态良好。随后将有更多的F1代CD163基因编辑猪陆续出生。【结论】制备的无抗性筛选标记的CD163双等位基因编辑猪,能够安全并快速地为培育抗PRRSV新品种猪提供育种材料。

【关键词】 CD163; CRISPR/Cas9; 猪; 猪繁殖与呼吸综合征;

【基金资助】 转基因生物新品种培育重大专项(2016ZX08010-004) 中国农业科学院科技创新工程(ASTIP-IAS05)

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This Article

ISSN:0578-1752

CN: 11-1328/S

Vol 51, No. 04, Pages 770-777

February 2018

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摘要

  • 0 引言
  • 1 材料与方法
  • 2 结果
  • 3 讨论
  • 4 结论
  • 参考文献