Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

Masakiyo Sakaguchi,Hiromi Kumon,Rie Kinoshita,Masami Watanabe,Hitoshi Murata,Yasutomo Nasu,Hideo Ueki,Haruki Kaku,Junichiro Futami,Nam-Ho Huh,Peng Huang,Yusuke Inoue,Shun-Ai Li,Endy Widya Putranto,I Made Winarsa Ruma

doi:10.1007/s12033-014-9738-0

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.

Highlights

In many biomedical studies, high-level expression of a gene in mammalian cells is a prime issue
In our previous study in which we tried to express two cDNAs with respective promoters in one construct, we unexpectedly found that the presence of a promoter at the 30-side of the cDNA significantly enhanced the efficiency of expression
A quantitative RT-PCR analysis showed that a detectable amount (7.35 ± 3.25) of transcripts containing KLF16 flanked by the 30-CMV promoter was observed when CMV/AS primer (Fig. 2b) was used for reverse transcription in cells transfected with the CMV–CMV vector (Table 1)

Summary

Introduction

High-level expression of a gene in mammalian cells is a prime issue. A variety of strong promoters have been exploited with the aim of high-level gene expression. Promoters of human elongation factor-1 alpha (EF-1a) [4] and b-actin [5, 6] are constitutively active in a broad range of cell types. Both promoters are often active in cells in which viral promoters fail to express downstream genes and in cells in which the viral promoters are gradually silenced as observed in embryonic stem cells [7]

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