SEMLIKI FOREST VIRUS-BASED DNA EXPRESSION VECTOR - TRANSIENT PROTEIN-PRODUCTION FOLLOWED BY CELL-DEATH

We have constructed a novel DNA expression vector based on Semliki For est virus (SFV). SFV produces nonstructural proteins (nsPs) which repl icate genomic RNA and amplify the mRNA encoding the structural protein s of SFV. A recombinant cDNA genome of SFV, in which the SFV structura l genes were replaced by a polylinker cassette to allow for insertion of heterologous DNA, was placed under the control of a cytomegalovirus immediate-early enhancer/promoter with a polyadenylation signal. Tran sfection of mammalian cells with this SFV-based plasmid vector, pSFV3- CMV-lacZ-pA, resulted in transient high-level expression of a beta-gal actosidase reporter gene. The expression level of beta-galactosidase f rom pSFV3-CMV-lacZ-pA was more than 20-fold higher than that obtained from the plasmid with deleted nsPs genes were essential for the high l evel of expression. Substantial beta-galactosidase activity was detect ed in the medium of pSFV3-CMV-lacZ-pA-transfected cells, suggesting th at the overproduction of beta-galactosidase caused cell death and rele ase of the protein into the medium. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from pSFV3-CMV-lac Z-pA constructed using an SFV replication system.