Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

Abstract

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Figure 1

Effect of different promoter on transfection efficiency and transient transgene expression. The pIRES-mediated vectors containing CMV, CAG, CHEF-1α, CMV mutant, HEF1-α, mouse CMV, CAG and PGK were transfected into CHO cells, and CHO cells were cultured in absence of G418 selection pressure for 48 h. (A) The eGFP of cells fluorescence profile was observed under fluorescence microscope; (B) The transfection efficiency were obtained using eGFP antibody analysis. (C) eGFP proteins transient expression levels. CMV promoter was regarded as 100, the MFI of other promoter were calculated. CMV, Cytomegalovirus major immediate-early; CAG, the CMV enhancer fused to the chicken beta-actin promoter; CHEF-1α, Chinese hamster elongation factor-1α; mouse CMV, mouse cytomegalovirus; HEF-1α, human elongation factor-1α; PGK, phosphoglycerate kinase; and CMV protein mutant, CAG enhancer. EPO, erythropoietin; SpA, simian virus 40 early polyadenylation signal; eGFP, enhanced green fluorescence protein.

Figure 2

Cells were collected at 10 generation post-transfection and the eGFP MFI was measured by flow cytometry. (A) The stably transfected cells were screened in medium containing G418 (800 μg/mL). The eGFP MFI of stably transfected cell lines containing different promoters were detected. Black bar represent the results from after 10 generations analyzed by flow cytometry. (B) Fold statistical analysis results of expression level, and the eGFP MFI was normalized to CMV promoter. Three stably transfected pools were generated for each vector. Cells were collected and measured for the eGFP MFI with the FACS Calibur (*P < 0.05, **P < 0.01).

Figure 3

Recombinant expression at mRNA level in cells transfected with CMV, CAG, HEF-1α, and CHEF-1α promoters at 10 generations post-transfection. (A) Target gene (eGFP) and internal reference gene (GAPDH) were measured by qRT-PCR. (B) The mRNA expression levels of cells were calculated using percentage of eGFP/GAPDH qRT-PCR values. qRT-PCR results were obtained three independent measurements.

Figure 4

The stability of eGFP expression in transfected CHO cells grown in the presence G418 selection pressure or absence of G418. (A) The stably transfected cells were passaged until 60 generation in the presence of G418 selection pressure (B) or in the absence of G418 selection pressure. The intensity of eGFP of cells were detected by flow cytometry at the passage 0, 10, 20, 30,40, 50, 60, respectively. The experiments were performed in triplicates. (C) The eGFP expression retention was calculated as the ratio of the MFI at the 60 generations stability testing to the MFI at the transient expression MFI testing. Retention of eGFP expression levels in cells transfected with CMV, CAG, CHEF-1α, CMV mutant, HEF1-α, mouse CMV, and CAG enhancer promoter-containing vectors (n = 3).

Figure 5

Gene copies per genome as determined by qPCR analysis. The gene copy number of CHO cells that stably transfected with CMV, CAG, CHEF-1α, HEF1-α element-containing vector was detected using qPCR at 10 generations post-transfection. (A) Thirty single stably clones was picked out and the gene copy number were analyzed by qPCR to detected the relationship between gene copy number. (B) The cells transfected with CMV promoter-containing vector was considered as 1.0. The relative copy number of the CAG, CHEF-1α, HEF1-α gene was calculated (*P < 0.05).

Figure 6

Analysis of EPO protein. The vectors containing EPO were transfected into CHO cells, and the stably transfected cells were screened under G418 selective pressure and thirty single cell clones were picked out. Cells supernatant were collected for analysis by ELISA to determine volumetric EPO production (mg/L). (A) CHEF-1α-EPO-containinng vector; (B) CMV-EPO-containing vector; (C) Western blot analysis. Lane 1, Un-transfected cells; Lane 2, CMV promoter; 3, CHEF1-α promoter, 4, Positive control.

Figure 7

Schematic representation of vectors containing different promters and the length of different elements. (A) The vectors construction that containing different element. (B) The lengths of the six different promoters, and CMV mutant, CAG enhancer.