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.

Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3′-side of the transgene

“…1A. pIDT-SMART (C-TSC) was previously reported (7). A 189-base pair element of the hTERT promoter (T) [accession no.…”

“…In order to improve the Ad-C-TSC-REIC vector and make it more permissive in donor HEK293 cells, without losing its high expression level in cancer cells, the present study modified the 3'-TSC (phTERT-pSV40-pCMV) region in the C-TSC cassette, as the 3'-region is known to affect gene expression level in vectors (7). To further simplify and weaken the 3'-TSC, the cancer-specific hTERT promoter (phTERT:T) was selected among the components of the 3'-TSC construct, since either pSV40 or pCMV was expected to demonstrate higher promoter activity compared with that of phTERT, based on previous studies (7,8).…”

“…To further simplify and weaken the 3'-TSC, the cancer-specific hTERT promoter (phTERT:T) was selected among the components of the 3'-TSC construct, since either pSV40 or pCMV was expected to demonstrate higher promoter activity compared with that of phTERT, based on previous studies (7,8). The present study first aimed to make a series of improved constructs using phTERT as the basis of the C-TSC vector structure.…”

“…To obtain improved therapeutic effects of Ad-REIC against cancer, our group previously developed a powerful gene expression system; the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 (SV40) and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal], which exhibited a higher efficacy of REIC/Dkk-3 expression compared with that of conventional vector systems (7,8). Adaptation of the C-TSC cassette and the cargo REIC/Dkk-3 cDNA to an adenovirus vector resulted in Ad-C-TSC-REIC, which induced apoptotic cell death to a greater extent in more types of cancer cells than the usual CMV promoter-driven Ad-REIC (7,8).…”

“…Adaptation of the C-TSC cassette and the cargo REIC/Dkk-3 cDNA to an adenovirus vector resulted in Ad-C-TSC-REIC, which induced apoptotic cell death to a greater extent in more types of cancer cells than the usual CMV promoter-driven Ad-REIC (7,8). However, due to the higher efficiency of virus-mediated REIC/Dkk-3 expression levels by Ad-C-TSC-REIC, we have identified that the viral production rate is markedly mitigated in HEK293 cells, which are commonly used in adenovirus expansion as a production donor.…”