Establishment of a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in gene using CRISPR/Cas9 technique.
10.12122/j.issn.1673-4254.2019.03.10
- Author:
Yanxia ZHOU
1
;
Weiwei HUI
1
;
Hongyang ZHANG
1
;
Lin ZOU
1
;
Penghui ZHANG
1
Author Information
1. Center for Clinical Examination, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, Chongqing 400014, China.
- Publication Type:Journal Article
- Keywords:
CRISPR/Cas9;
G6PD;
HEK293T cells;
gene knockout
- MeSH:
CRISPR-Cas Systems;
HEK293 Cells;
Humans;
Mutation;
Plasmids;
RNA, Guide
- From:
Journal of Southern Medical University
2019;39(3):320-327
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To establish a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in gene using CRISPR/Cas9 technique.
METHODS:We designed 4 pairs of small guide RNA (sgRNA) for c.392G>T(p.131G>V) mutation site, and constructed exogenous PX458 plasmids expressing Cas9-sgRNA. The plasmids were transfected into HEK293T cells, and the cells expressing GFP fluorescent protein were separated by flow cytometry for further culture. After verification of the knockout efficiency using T7 endonuclease Ⅰ, the monoclonal cells were screened by limiting dilution and DNA sequencing to confirm the knockout. We detected the expressions of mRNA and protein and examined functional changes of the genetically modified cells.
RESULTS:We successfully constructed the Cas9-sgRNA exogenous PX458 plasmid based on the c.392G>T(p.131G>V) mutation site of gene. The editing efficiency of the 4 pairs of sgRNA, as detected by T7E1 enzyme digestion, was 6.74%, 12.36%, 12.54% and 2.94%. Sanger sequencing confirmed that the HEK293T cell line with stable knockout of c.392G>T(p.131G>V) was successfully constructed. The genetically modified cells expressed lower levels of mRNA and protein and showed reduced enzyme activity and proliferative capacity and increased apoptosis in response to vitamin K3 treatment.
CONCLUSIONS:We successfully constructed a stable HEK293T cell model with gene c.392G>T(p.131G>V) mutation site knockout to facilitate future study of gene repair.
