Construction and application of 293T-CymR cell strain
10.13200/j.cnki.cjb.004634
- VernacularTitle:293T-CymR细胞株的构建及其应用
- Author:
Mengjun WANG
1
Author Information
1. Viral Vaccine Research Laboratory, Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, Hubei Province, China
- Publication Type:Journal Article
- Keywords:
Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9;
Cysteine metabolism regulator(Cy-mR);
HEK293T cells;
Cumate gene-switch;
Cell strain construction
- From:
Chinese Journal of Biologicals
2026;39(01):7-16
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a 293T-CymR cell strain to express cysteine metabolism regulator(CymR) component by gene editing technology, and to regulate the target gene expression with CuO regulatory sequence promoter.Methods Based on clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 technology, the intracellular β factor receptor, C-C chemokine receptor(CCR5) gene, one of the safe harbors in HEK293T cell genome, was chosen as the target site for inserting the desired gene. The selection gene Pac(denoted as PuroR) encoding puromycin N-acetyltransferase was linked to the 3'end of the CymR gene via an internal ribosome entry site(IRES), sharing a cytomegalovirus(CMV) promoter. The 293 TCymR monoclonal cell strain capable of expressing CymR was screened by Puro resistance and limiting dilution method. The CymR gene position and protein function in the 293T-CymR monoclonal cell strain were verified by PCR, Western blot and fluorescence analysis.Results The pY93-CymR plasmid expressing both the target and resistance genes, was successfully constructed. Following co-transfection with the pX330-CCR5 plasmid into HEK293T cells, four 293T-CymR monoclonal cell strains were obtained via Puro resistance selection and the limiting dilution method. The PCR and Western blot analysis indicated that CymR gene was successfully inserted into the intended CCR5 site and CymR protein was efficiently produced in three of these clones. Subsequent transfection tests demonstrated that the produced CymR could successfully suppress the gene expression under the CMV promoter containing CuO element.Conclusion CymRgene was successfully inserted into the CCR5 locus of HEK293T cell genome, and 293T-CymR monoclonal cell strains capable of expressing CymR and regulating gene expression were obtained.
