Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells

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.

Figure 1

Schematic diagram of vectors pIRES-EGFP (A) and pIRES-EGFP1-10 (B) containing different size of IRES fragment. CMV, human cytomegalo virus immediate early gene promoter; EGFP, enhanced green fluorescent protein; IVS, synthetic intron; IRES, internal ribosome entry site.

Figure 2

Fluorescence profile of EGFP in CHO-S cells. CHO-S cells transfected with vectors containing IRES elements from different resources were measured using FACS after 48 h transfection. (A) Representative of high EGFP fluorescence profile in cells with the pIRES-EGFP-9. (B) Representative of medium EGFP fluorescence profile in cells with the pIRES-EGFP-4. (C) Representative of low EGFP fluorescence profile in cells with the pIRES-EGFP-2. (D) Representative of untransfected cells. Scale bar, 100 μm.

Figure 3

The EGFP protein levels in CHO-S cells determined by flow cytometry. Representative of EGFP fluorescence profile 48 h post-transfection (A) and stable transfection (B). Cells with pIRES-EGFP-2 represented of low EGFP protein level. Cells with pIRES-EGFP-4 and pIRES-EGFP-6, represented of medium EGFP protein level. Cells with pIRES-EGFP-9 represented of high EGFP protein level.

Figure 4

Relative EGFP protein expression levels in CHO-S cells. Transient EGFP expression was measured by flow cytometry 48 h post-transfection. All the experiments were repeated three times. It indicates that the EGFP expression levels from pIRES-EGFP-9 with the IRES of HRV were higher compared with those from other vectors (P < 0.05).

Figure 5

Representative positive colony in stably transfected CHO-S cells. Transfection and drug selection were carried out as stated in the portion of materials and methods. Single clones growing from each of the population were photographed with fluorescence microscope. Three stably single clone pools were chosen for vectors. EGFP fluorescence in cells stably transfected with pIRES-EGFP-9 (A) pIRES-EGFP-8 (B) and pIRES-EGFP-2 (C). (D) Image of colony under white light. Scale bar, 50 μm.

Figure 6

Relative EGFP expression levels and copy numbers in stably transfected CHO-S cells. Cells were tested for the MFI with the FACS Calibur (A) and copy number was measured by qPCR (B). It indicated that relative EGFP protein levels in stable transformation cells was decreased and cells transfected with pIRES-EGFP-9 with the IRES of HRV had the highest gene copy numbers.

Figure 7

Analysis of EPO protein expression by western bolt. The vectors pIRES-EPO-8 and pIRES-EPO-9 were transformed into CHO-S cells, and four clones of cells were chosen, respectively. Lane 1–4, pIRES-EPO-9; Lane 5–8, pIRES-EPO-8.