Dramatic increase in expression of a transgene by insertion of promoters downstream of the cargo gene

Abstract

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5' located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

Fig. 1

Schematic diagram of modified gene expression systems and their capabilities for gene expressions. a A series of indicated plasmids were constructed on the basis of the promoter-less pDNR-1r vector. b Expression of KLF16 protein was assessed by Western blot analysis after transfecting the indicated plasmids carrying KLF16 cDNA in HEK293, MCF7, PC-3, HeLa, and HepG2 cells. c Plasmid vectors carrying various cDNAs were constructed using the same series of vectors as those shown in (A). The vectors were transfected to HEK293 cells, and the level of each protein was determined by Western blot analysis. Lane numbers in b and c correspond to the vector numbers shown in (a)

Fig. 2

Orientation of the 3′-CMV promoter-independent gene expression. a Orientation of the 3′-CMV promoter in the CMV-KLF16-CMV construct was flipped, termed CMV-KLF16-CMV (flipped), and then expression of KLF16 protein was assessed by Western blot analysis after transfecting the plasmid including indicated comparative vectors in HEK293. Tubulin was used as a control for loaded amounts of protein. b Primers were designed to the indicated locations of the CMV-KLF16-CMV and the CMV-KLF16-CMV (flipped) constructs (see Materials and Methods)

Fig. 3

Construction of the C-TSC cassette. a Expression of KLF16 (top) or REIC/Dkk-3 (bottom) proteins after transfecting the indicated constructs including a vector containing the CAG promoter in HEK293 cells. Tubulin was used as a control for loaded amounts of protein. b To further improve the expression, 3 promoter sequences of hTERT, SV40, and CMV were tandemly inserted downstream of the REIC cDNA-BGH (bovine growth hormone)-polyA signal. RU5′, a part of the HTLV type 1 LTR inserted for better efficiency of transcription and translation. CMV–CMV and CMV–RU5′–hTERT–SV40–CMV were constructed on the basis of pDNR-1r and pIDT-SMART promoter-less vectors (top). The vectors were transfected to HEK293 cells, and levels of inserted REIC protein were determined by Western blot analysis (top). Tubulin was used as a control for loaded amounts of protein. Finally, we named the improved high expression cassette C-TSC (bottom)

Fig. 4

Expression levels of a secreted protein, erythropoietin (EPO). 24 h after transfection with the indicated vectors, 10 μl of serum-free culture medium and cellular protein extracts (10 μg) were subjected to electrophoresis, and EPO protein was detected by Western blot analysis. pTracer and pEF6 were from commercial source. b EPO was recovered from 1 liter of each culture medium of 293-F cells, electrophoresed, and stained with CBB (top). The amount of EPO obtained from 1 liter of each suspension culture (bottom). c The purified EPO was treated (+) or not treated (−) with PNGase F. d Effect of the purified EPO on growth of TF-1 cells was examined by an MTT assay

Fig. 5

Expression of REIC/Dkk-3 by an adenovirus vector having the C-TSC cassette. a Expression of REIC/Dkk-3 was determined in human cancer cell lines (PC-3, prostate adenocarcinoma cell line; HeLa, cervix adenocarcinoma cell line). Tubulin was used as a control for loaded amounts of protein. b Induction of apoptosis in human cancer cell lines (PC-3; KPK-1, renal clear cell carcinoma cell line) by an adenovirus vectors carrying REIC/Dkk-3 infected at 100 MOI. * p < 0.05