Synthetic enhancers including TFREs improve transgene expression in CHO cells

Abstract

The human cytomegalovirus major immediate early gene (CMV) promoter is currently the most preferred promoter for recombinant therapeutic proteins (RTPs) production in CHO cells. To enhance the production of RTPs, five synthetic enhancers including multiple transcription factor regulatory elements (TFREs) were evaluated to enhance recombinant protein level in transient and stably transfected CHO cells. Compared with the control, four elements can enhance the report genes expression under both two transfected states. Further, the function of these four enhancers on human serum albumin (HSA) were investigated. We found that the transient expression can increase by up to 1.5 times, and the stably expression can maximum increase by up to 2.14 times. The enhancement of transgene expression was caused by the boost of their corresponding mRNA levels. Transcriptomics analysis was performed and found that transcriptional activation and cell cycle regulation genes were involved. In conclusion, optimization of enhancers in the CMV promoter could increase the production yield of transgene in transfected CHO cells, which has significance for developing high-yield CHO cell expression system.

Keywords: CHO cell expression system; CMV promoter; Enhancer; Recombinant proteins; Synthetic promoter.

Fig. 1

Schematic representation of the different expression vectors. A: The original pEGFP, pSEAP, pHSA vector. B: Truncated promoter vectors. C: Expression vectors containing different synthetic enhancers for expression of reporter genes: D: Expression vectors containing different synthetic enhancers for expression of human serum albumin. pCMV: CMV promoter; mini CMV: Truncated CMV promoter; E1, E2, E3, E4, E5 are different synthetic enhancers.

Fig. 2

Effect of different synthetic enhancers on the transient expression of reporter genes. A: The fluorescence microscopy images at 48 h post-transfection. B: Viable cell density of EGFP in CHO cells. C: Viable cell density of SEAP in CHO cells. D: Median fluorescence intensity of EGFP. E: Transient expression of SEAP. Compared to control, *P < 0.05, **P < 0.005. ***P < 0.0001.

Fig. 3

Effect of different synthetic enhancers on the stable expression of reporter genes. A: Fluorescence microscope of a stable cell pool. B: Viable cell density of EGFP stable expression. C:Viable cell density of SEAP stable expression. D: Western blot results. E: Grey-scale analysis of EGFP Western blot results. F: Stable expression of SEAP.

Fig. 4

Effect of different synthetic enhancers on the transient expression of human serum albumin A:Viable cell density; B:Western blot results; C:Grey-scale analysis of human serum albumin Western blot results;

Fig. 5

Effect of different synthetic enhancers on the stable expression of human serum albumin. A: Viable cell density; B: Western blot results; C: Grey-scale analysis of human serum albumin Western blot;

Fig. 6

Analysis of mRNA relative expression levels in HSA stably cell pools. A: mRNA expression levels in HSA stable cell pools; B: The correlation between HSA expression level and mRNA level was evaluated using a correlation coefficient (R2). Compared to control (pHSA), *P < 0.05. **P < 0.005.

Fig. 7

Analysis of gene expression differences between high expression and control cell lines. A: Differential gene heatmap; B: Expression correlation scatter plot: Group 2 and Group 1 represents highly expressed strain and control cell line, respectively.