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.
Highlights
T In the present study, six commonly used promoters, including cytomegalovirus major immediateearly (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation
The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; all elements were transfected into Chinese hamster ovary (CHO) cells
At 48 h post-transfection, the fluorescence intensity of CHO cells transfected with different types of promoter was observed under a fluorescence microscope (Fig. 1A)
Summary
T In the present study, six commonly used promoters, including cytomegalovirus major immediateearly (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation. R 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. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; all elements were transfected into CHO cells. Transfected CHO cells were identified via screening under the selection pressure of G418. The CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV. E 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
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