PHAGE RNA-POLYMERASE VECTORS THAT ALLOW EFFICIENT GENE-EXPRESSION IN BOTH PROKARYOTIC AND EUKARYOTIC CELLS

We have developed expression vectors that direct the synthesis of prot eins from a common set of signals in both prokaryotic and eukaryotic c ells. To allow transcription from a common promoter the vectors rely u pon a phage RNA polymerase (RNAP). To direct initiation of translation to the same start codon the vectors utilize an internal ribosome entr y site (IRES) from encephalomyocarditis virus (EMCV) that has been mod ified to include a prokaryotic ribosome-binding site (RES) at an appro priate distance upstream from the desired start codon. These vectors p rovide levels of expression in eukaryotic cells that exceed those of a conventional RNAP-II-based system by 7-fold, and expression in bacter ial cells at levels comparable to other phage RNAP-based systems. Incl usion of a Inc repressor and a phage promoter/lac operator fusion elem ent allows tight regulation. Cotransfection of eukaryotic cells with t he expression vector and a vector that encodes the phage RNAP provides high-level transient expression without the need to construct special ized stable cell lines.