CRISPR-Cas9-based site-directed knock-in of VEGF165 gene in a HEK293T cell

Zaiyu Guo, Heliang Zhang, Qian Chen, Yanwei Hou, Taotao Shui, Lili Wu, Yijie Liu, Qiaoman Fei, Huan Huang, Lei Lei, Yan Sun, Yu Kong, Xiujuan Zhao, Yating Han, Bing Yang, Ling Zhang
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Abstract

Objective To construct a human renal epithelial cell line HEK293T by CRISPR-Cas9-based site-directed knock-in of vascular endothelial growth factor 165 (VEGF165) gene, and avoid the off-target effect caused by lentivirus infection. Methods The VEGF165 expression vector with homologous arm (pUCm-T-VEGF165 plasmid) and the sgRNA expression vector [pSpCas9(BB)-2A-Puro-sgRNA plasmid] were designed and constructed based on the DNA sequence of the EZH2 gene, and then co-transfected into HEK293T cells. The expression of VEGF165 mRNA was detected by qPCR and the expressions of VEGF165 proteins were detected by Western Blot. Results The qPCR and Western Blot results showed that, comparing with the control, the pUCm-T-VEGF165 plasmid and pSpCas9(BB)-2A-Puro-sgRNA plasmid, the expression of the co-transfection plasmid were significantly increased, i.e. 3.42±0.30 vs. 1.02±0.21, 1.13±0.16 and 0.98±0.18 for the VEGF165 mRNA level (all P<0.01), and 1.13±0.16 vs. 1.02±0.06, 0.88±0.03 and 0.80±0.05 for the VEGF165 protein level (all P<0.01), respectively. Besides, the expression of EZH2 was significantly down-regulated, i.e. 0.14±0.06 vs. 1.08±0.11, 1.02±0.12 and 1.13±0.16 for the EZH2 mRNA level (all P<0.01), and 0.23±0.03 vs. 1.05±0.13, 0.91±0.04 and 0.81±0.06 for the EZH2 protein level (all P<0.01), respectively. This result showed that the VEGF165 was successfully inserted into the EZH2 genome, interfering the EZH2 expression. Conclusions VEGF165 gene can be successfully knocked into HEK293T cells by CRISPR/Cas9 system. Key words: Vascular endothelial growth factor 165; CRISPR/Cas9; HEK293T cells
HEK293T细胞中VEGF165基因的CRISPR-Cas9位点定向敲除
目的利用CRISPR-Cas9位点定向敲除血管内皮生长因子165(VEGF165)基因构建人肾上皮细胞系HEK293T,避免慢病毒感染引起的脱靶效应。方法根据EZH2基因的DNA序列,设计构建具有同源臂的VEGF165表达载体(pUCm-T-VEGF165质粒)和sgRNA表达载体(pSpCas9(BB)-2A-Puro-sgRNA质粒),并将其共转染HEK293T细胞。qPCR法检测VEGF165 mRNA的表达,Western Blot法检测VEGF1 65蛋白的表达。结果qPCR和Western Blot结果显示,与对照、pUCm-T-VEGF165质粒和pSpCas9(BB)-2A Puro-sgRNA质粒相比,共转染质粒的表达显著增加,即VEGF165 mRNA水平分别为3.42±0.30和1.02±0.21、1.13±0.16和0.98±0.18(均P<0.01),VEGF165蛋白水平分别为0.88±0.03和0.80±0.05(均P<0.01)。此外,EZH2的表达显著下调,即EZH2 mRNA水平分别为0.14±0.06对1.08±0.11、1.02±0.12和1.13±0.16(均P<0.01),EZH2蛋白水平分别为0.23±0.03对1.05±0.13、0.91±0.04和0.81±0.06(均<0.01)。这一结果表明VEGF165成功地插入EZH2基因组,干扰了EZH2的表达。结论利用CRISPR/Cas9系统可以成功地将VEGF165基因敲除到HEK293T细胞中。关键词:血管内皮生长因子165;CRISPR/Cas9;HEK293T细胞
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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