CRISPR/Cas9 Nuclease-Mediated Gene Knock-In in Bovine-Induced Pluripotent Cells
CRISPR/Cas9 Nuclease-Mediated Gene Knock-In in Bovine-Induced Pluripotent Cells
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DOI：
10.1089/scd.2014.0278
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发表时间：
2015-02-01
影响因子：
4
通讯作者：
Kim, Jongpil
中科院分区：
文献类型：
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作者：
Heo, Young Tae;Quan, Xiaoyuan;Kim, Jongpil
在家畜（如牛）中进行有效和精确的基因工程在农业和生物医学方面具有很大的前景。然而，产生多能干细胞的技术以及用于家畜基因靶向的可靠工具仍然效率低下，因此没有常规使用。在这里，我们报告了使用牛多能细胞和成簇规则间隔短回文重复序列（CRISPR）/Cas9核酸酶在牛基因组中的高效基因靶向。首先，我们通过山中因子的异位表达和GSK 3 β和MEK抑制剂（2 i）处理从牛体细胞成纤维细胞产生诱导多能干细胞（iPSC）。我们观察到这些牛iPSC在畸胎瘤中的基因表达和发育潜力方面与幼稚多能干细胞高度相似。此外，对牛NANOG基因座具有特异性的CRISPR/Cas9核酸酶在牛iPSC和胚胎中显示出对牛基因组的高效编辑。综上所述，CRISPR/Cas9核酸酶介导的同源重组靶向牛多能细胞是一种有效的基因编辑方法，未来可用于产生转基因家畜。
Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3 beta and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naive pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.