Evaluation of the Promoter Activity of the CHO Cell Expression System Based on Site-specific Integration
10.13865/j.cnki.cjbmb.2024.09.1131
- VernacularTitle:基于位点特异性整合评价CHO细胞表达系统启动子活性的研究
- Author:
Chen LU
1
;
Zi-Yu WANG
;
Yan-Fei CAI
;
Yong-Qiang DENG
;
Jian JIN
;
Xue-Feng DING
;
Yun CHEN
Author Information
1. 江南大学生命科学与健康工程学院,江苏无锡 214122
- Keywords:
promoter screening;
site-specific integration;
Chinese hamster ovary cells(CHO);
CRISPR/Cas9
- From:
Chinese Journal of Biochemistry and Molecular Biology
2024;40(10):1400-1408
- CountryChina
- Language:Chinese
-
Abstract:
In industrial production,the expression level of drug proteins in Chinese hamster ovary cells(CHO)is influenced by many factors:the regulatory elements on transcription and translation,the ge-nomic integration sites,and the expression system.Transcription,as the first step of gene expression,largely affects protein expression,and the promoter plays a crucial role in the initiation of transcription.Most of the promoters were screened through transient transfection or random integration,but the presence of unclear copy number or random integration sites makes it difficult to accurately evaluate the promoter activity.To some extent,site-specific integration can reduce the impact of positional effects on exogenous genes and may potentially increase the expression level of exogenous genes.In the early stage of our re-search,multiple sites that can stably express exogenous proteins were identified and verified in the CHO cell genome.In this study,one of these sites(2c6)was selected for the evaluation of promoter activity.The CRISPR/Cas9 gene editing technique was used to site-specifically integrate the reporter gene(EG-FP)regulated by the simian virus early promoter(SV40),mouse elongation factor-1α(mEF-1α),chicken β-actin(cACTB)promoter,and human phosphoglycerate kinase promoter(hPGK)into the 2c6 site,respectively.The mean fluorescence intensity of the cells was analyzed by flow cytometry,and the mRNA level of EGFP was detected by qPCR to comprehensively evaluate the activity of the promoter.The results showed that the activities of the mEF-1α and mACTB promoters were better than those of SV40 and hPGK.The results of the secondary flow cytometry sorting showed that site-specific integration can more accurately evaluate the activity of the promoter in the CHO cell expression system.
