Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells

Abstract

We have developed expression vectors that direct the synthesis of proteins from a common set of signals in both prokaryotic and eukaryotic cells. To allow transcription from a common promoter the vectors rely upon a phage RNA polymerase (RNAP). To direct initiation of translation to the same start codon the vectors utilize an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) that has been modified to include a prokaryotic ribosome-binding site (RBS) at an appropriate distance upstream from the desired start codon. These vectors provide levels of expression in eukaryotic cells that exceed those of a conventional RNAP-II-based system by 7-fold, and expression in bacterial cells at levels comparable to other phage RNAP-based systems. Inclusion of a lac repressor and a phage promoter/ lac operator fusion element allows tight regulation. Cotransfection of eukaryotic cells with the expression vector and a vector that encodes the phage RNAP provides high-level transient expression without the need to construct specialized stable cell lines.