CRISPR-Cas9-based site-directed knock-in of VEGF165 gene in a HEK293T cell
10.3760/cma.j.issn.1673-4181.2019.01.007
- VernacularTitle:采用CRISPR knockin技术实现VEGF165定点敲入HEK293T细胞系的构建
- Author:
Zaiyu GUO
1
;
Heliang ZHANG
;
Qian CHEN
;
Yanwei HOU
;
Tao SHUI
;
Lili WU
;
Yijie LIU
;
Qiaoman FEI
;
Huan HUANG
;
Lei LEI
;
Yan SUN
;
Yu KONG
;
Xiujuan ZHAO
;
Yating HAN
;
Bing YANG
;
Ling ZHANG
Author Information
1. 天津泰达医院神经外科 300457
- Keywords:
Vascular endothelial growth factor 165;
CRISPR/Cas9;
HEK293T cells
- From:
International Journal of Biomedical Engineering
2019;42(1):39-44
- CountryChina
- Language:Chinese
-
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.
