CRISPR/Cas9 Nuclease-Mediated Gene Knock-In in Bovine-Induced Pluripotent Cells

被引:63
作者
Heo, Young Tae [1 ]
Quan, Xiaoyuan [2 ]
Xu, Yong Nan [1 ,3 ]
Baek, Soonbong [2 ]
Choi, Hwan [2 ]
Kim, Nam-Hyung [1 ]
Kim, Jongpil [2 ]
机构
[1] Chungbuk Natl Univ, Dept Anim Sci, Ctr Anim Bioreactor & Xenotransplantat, Cheongju, South Korea
[2] Dongguk Univ, Dept Biomed Engn, Lab Stem Cells & Cell Reprogramming, Seoul 100715, South Korea
[3] Yanbian Univ, Dept Vet Med, Coll Agr, Yanji, Jilin Province, Peoples R China
基金
新加坡国家研究基金会;
关键词
EMBRYONIC STEM-CELLS; TRANSGENIC LIVESTOCK; CRISPR-CAS9; SYSTEM; DEFINED FACTORS; GROUND-STATE; FIBROBLASTS; GENERATION; INDUCTION; FUTURE; NANOG;
D O I
10.1089/scd.2014.0278
中图分类号
Q813 [细胞工程];
学科分类号
摘要
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.
引用
收藏
页码:393 / 402
页数:10
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