Abstract
Chinese hamster ovary (CHO) cells are mainly used for recombinant protein production. However, the unstable transgene expression and lower transgene copy numbers are the major issues need to be resolved. Here, eleven internal ribosome entry site (IRES) elements from viral and cellular IRES were evaluated for foreign gene expression in CHO-S cells. We constructed eleven fusing plasmids containing different IRES sequences downstream of the enhanced green fluorescent protein (EGFP) gene. EGFP expression was detected by flow cytometry and the transgene copy number was evaluated by quantitative PCR. The erythropoietin (EPO) protein was also used to assess the stronger IRES. The results showed that IRES from human rhinovirus (HRV) exhibited the highest EGFP expression level under transient and stable transfections. The EGFP expression level of vector with IRES from HRV was related to the gene copy number in stably transfected CHO-S cells. Moreover, IRES from HRV induced higher expression level of EPO compared with one mutant IRES from EMCV in transfected cells. In conclusion, IRES from HRV can function as a strong IRES element for stable expression in CHO-S cells, which could potentially guide more effective foreign gene expression in CHO-S cells.
Highlights
Mammalian cell expression system is the most essential technique for the establishment of the production platform of therapeutic recombinant protein
The enhanced green fluorescent protein (EGFP) expression level was determined by subjecting to FACS analysis(Figs 3A, 4) when Chinese hamster ovary (CHO) cells were reached to 60–70% confluence
Group 1 with high expression represented by pIRES-EGFP-9 (Fig. 2A) and pIRES-EGFP-8; group 2 with medium expression represented by pIRES-EGFP-4 (Fig. 2B) and pIRES-EGFP-6; group 3 with low expression represented by pIRES-EGFP-2 (Fig. 2C) and pIRES-EGFP-7
Summary
Mammalian cell expression system is the most essential technique for the establishment of the production platform of therapeutic recombinant protein. Foreign gene expression in eucaryotic organism is regulated by complex events including DNA transcription, RNA translation, proteins post-translational modification, and secretion[5]. IRES sequences of Encephalomyocarditis virus (EMCV) to connect two independent genes transcribed from the same promoter within the single expression vectors had been used in gene therapy[6]. The varied IRES elements from viral and cellular IRES including immunoglobulin heavy chain binding protein (BIP), cationic amino acid transporter 1 (CAT-1), c-myc, Hepatitis C (HCV), vascular endothelial growth factor (VEGF) and type[1] collagen inducible protein (VCIP), apoptotic protease activating factor 1 (Apaf-1), Encephalomyocarditis virus (EMCV), Human rhinovirus (HRV) and NF-kappa B repressing factor (NRF) were tested in the transgene stability and the transgenic expression level in CHO cells. The results found here will be useful for designing optimal bicistronic vectors with long-term transgenic stability and high expression ability for diverse CHO cell engineering application
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