Generation of an EFNB2-2A-mCherry reporter human embryonic stem cell line using CRISPR/Cas9-mediated site-specific homologous recombination
Generation of an EFNB2-2A-mCherry reporter human embryonic stem cell line using CRISPR/Cas9-mediated site-specific homologous recombination
复制标题
使用 CRISPR/Cas9 介导的位点特异性同源重组生成 EFNB2-2A-mCherry 报告基因人胚胎干细胞系
DOI：
10.1016/j.scr.2021.102241
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发表时间：
2021
影响因子：
1.2
通讯作者：
Wei Lei
中科院分区：
文献类型：
--
作者：
Ying Huang;Hongchun Wu;Xinglong Han;Jie Wu;Miao Yu;Zhen-ao Zhao;Zhenya Shen;Shijun Hu;Wei Lei
Ephrin B2（EFNB 2）是第一个被鉴定的和最广泛使用的动脉内皮细胞（AECs）的标记物。我们通过CRISPR/Cas9介导的将2A-mCherry盒插入EFNB 2基因位点，紧接在翻译终止密码子之前，产生了杂合EFNB 2 - 2A-mCherry报告基因H1细胞系H1-EFNB 2 - 2A-mCherry+/−（WAe 001-A-57）。H1-EFNB 2 -2A-mCherry报告细胞是多能的，并且可以分化成所有三种胚层谱系。在内皮细胞分化过程中，当EFNB 2表达增加时，观察到mCherry的同时表达。因此，所产生的报告细胞使得能够活体鉴定EFNB 2阳性AEC，并筛选促进AEC分化的小分子化合物和靶基因。
Ephrin B2 (EFNB2) is the first identified and most widely used marker for arterial endothelial cells (AECs). We generated a heterozygous EFNB2-2A-mCherry reporter H1 cell line, H1-EFNB2-2A-mCherry+/−(WAe001-A-57), by CRISPR/Cas9-mediated insertion of 2A-mCherry cassette into theEFNB2gene locus, immediately before the translation stop codon. The H1-EFNB2-2A-mCherry reporter cells were pluripotent and could differentiate into all three germ layer lineages. Simultaneous expression ofmCherrywas observed when expression ofEFNB2was increased during endothelial cell differentiation. Thus, the generated reporter cells enable live identification of EFNB2-positive AECs, and screening of small molecule compound and target genes that promote AEC differentiation.