The EF-1α core promoter has higher expression efficiency.
First, we evaluated four promoters commonly used in expression vectors to determine their ability to transiently express genes in different kinds of cells. 24 hours after transfection, the fluorescence of eGFP was observed using an inverted fluorescence microscope. The fluorescent images showed that the protein expression levels regulated by four promoters were different (Fig. 2A and 2B). Then the cells were collected and the expression of eGFP protein was detected by western blot analysis (Fig. 2C and 2D). The results show that the eGFP expression levels from western blot assays are consistent with those observed by fluorescence microscopy. The vector containing EF-1α core promoter showed the highest eGFP expression levels in three species of cells and the Polr2a promoter and PGK promoter have different intensity on eGFP expression in different cell species. The EF-1α core promoter was therefore retained and used in combination with different enhancers to detect the effect on reporter gene expression.
The combination of CMV enhancer and EF-1α core promoter increased reporter gene expression.
The SV40 enhancer was used to replace the CMV enhancer in vector 3, and the reporter gene expression strength was compared between these two different enhancer-promoter combinations and the pcDNA3-eGFP vector. The fluorescence of eGFP showed that the CMV enhancer combination with EF-1α core promoter can better enhance reporter gene expression (Fig. 3A and 3B). Western blot detection showed consistent results with fluorescence (Fig. 3C and 3D).
Intron incorporation into the combination of CMV enhancer and EF-1α core promoter cannot help gene expression.
Numerous studies have shown that adding heterologous introns to expression vectors increases gene expression (Yew, Wysokenski et al. 1997, Baier, Jacobebbinghaus et al. 2020). Therefore, we inserted two different introns into downstream of the EF-1α core promoter in vector 3 to determine whether current combinations of regulatory elements could further enhance transgene expression. The expression of reporter gene was detected by fluorescence (Fig. 4A and 4B) and western blot (Fig. 4C, and 4D). Interestingly, hybrid intron and EF-1α intron A, which were originally reported to have the function of up-regulating gene expression (Melcher, Grosch et al. 2002, Zhang, Zha et al. 2016), instead of working as expected, showed significant inhibition of eGFP gene expression in L929 and MARC145 cells after combination with the CMV enhancer and EF-1α core promoter.
The combination of EF-1α core promoter and EF-1α intron A increased protein expression.
Because incorporating intron into the combination of CMV enhancer and EF-1α core promoter didn't show the upregulation of gene expression, we constructed vectors 7 and 8 by removing the CMV enhancer from vectors 5 and 6 by deletion mutation. These vectors were then transfected into HeLa, L929 and MARC145 cells, and the expression of the eGFP gene was detected by fluorescence observation and western blotting. The results showed that the combination of hybrid intron and EF-1α core promoter did not upregulate reporter gene expression after the removal of the CMV enhancer, but the EF-1α intron A exhibits a synergistic effect with the EF-1α core promoter to enhance gene expression (Fig. 5A and 5B). The result of western blot indicates that the cooperation of EF-1α core promoter and EF-1α intron A increased the eGFP transgene expression by 2.49-fold, 2.36-fold and 2.31-fold than pcDNA3-eGFP vector before modification in HeLa, L929 and MARC145(Fig. 5C and 5D).
The combination of EF-1α core promoter, EF-1α intron and terminators failed to further improve protein expression.
Specific combinations of terminators and promoters has been widely demonstrated to enhance gene expression in human, plant cells, and yeast (West and Proudfoot 2009, Curran, Karim et al. 2013, Diamos and Mason 2018, Ramakrishnan, Prattipati et al. 2020). We thus select two terminators commonly used in expression vectors, the CYC1 terminator and the TEF terminator, inserted into downstream of the eGFP sequence of vector 8 to form vectors 9 and 10. We then compared the expression of the eGFP of the two newly constructed vectors with vector 8 and pcDNA3-eGFP. The results showed that the expression of eGFP protein did not increase with the addition of the terminator, and the vector inserted with the CYC1 terminator was even significantly fewer able to express eGFP protein than before(Fig. 6A、6B、6C and 6D).
Vector elements composed by TEF terminator with CMV enhancer, EF-1α core promoter can improve protein expression.
Because the combination of EF-1α core promoter, EF-1α intron and terminators didn’t increase the reporter gene expression, we thus tried to combination terminators with alternative regulatory elements. The CYC1 terminator and TEF terminator are inserted downstream of the eGFP sequence in vector 3 to construct vectors 11 and 12, respectively, forming a combination of terminators with the CMV enhancer, EF-1α core promoter. Subsequently, we detected the effect of combinations of altered expression elements on the expression of the reporter genes by fluorescence intensity and western blotting. It was found that the expression of the eGFP protein was significantly increased by the insertion of the TEF terminator in vector11 compared to pcDNA3-eGFP and vector3, while the expression of the gene was reduced by the insertion of the CYC1 terminator. (Fig. 7A and 7B). The combination of vector elements formed by TEF terminator, CMV enhancer and EF-1α core promoter is the combination of vector elements obtained in this experiment that promotes eGFP protein expression intensity the most, the western blot results showed that with this combination, the expression of the eGFP protein in HeLa, L929, and MARC145 cells increased by a factor of 2.84, 3.09, and 3.21 respectively, compared to pcDNA3-eGFP (Fig. 7C and 7D).