A SELF-INITIATING EUKARYOTIC TRANSIENT GENE-EXPRESSION SYSTEM BASED ON COTRANSFECTION OF BACTERIOPHAGE-T7 RNA-POLYMERASE AND DNA VECTORS CONTAINING A T7-AUTOGENE

A novel cytoplasmic gene expression system has been developed. This sy stem differs from other expression systems in that it relies on the co -delivery of plasmid DNA and T7 RNA polymerase (RNAP) during transfect ion. The plasmid contains a T7 RNAP gene driven by the T7 promoter (T7 autogene) and a functional/reporter gene driven by another T7 promote r (T7T7/T7-gene construct). Once this DNA - enzyme complex is introduc ed into eukaryotic cells, the transcription of the T7 RNAP and the fun ctional/reporter genes is initiated by the cc-delivered T7 RNAP. The T 7 RNAP, which is responsible for the initiation and maintenance of exp ression of both T7 and functional/reporter genes, is replenished by tr anslation of newly synthesized T7 mRNA. This T7 system was designed in such a manner that the expression of the functional/reporter genes ca n occur in the cytoplasm and does not require any nuclear involvement. When transfected by either a pT7T7/T7Luc or a pT7T7/T7hGH plasmids wi th the cointroduced T7 RNAP, mouse L cells were found to express high levels of luciferase immediately after transfection, apparently due to the cytoplasmic gene expression; the expression of human growth hormo ne (hGH) could be sustained for at least 6 days. Both T7 and hGH mRNA were expressed by the cells transfected with pT7T7/T7hGH. These result s suggest that this cytoplasmic expression system may be used for cert ain targets of somatic gene therapy.