STRINGENT CHEMICAL AND THERMAL REGULATION OF RECOMBINANT GENE-EXPRESSION BY VACCINIA VIRUS VECTORS IN MAMMALIAN-CELLS

We developed a stringently regulated expression system for mammalian c ells that uses (i) the RNA polymerase, phi 10 promoter, and T phi tran scriptional terminator of bacteriophage T7; (ii) the lac repressor, la c operator, rho-independent transcriptional terminators and the gpt ge ne of Escherichia coil; (iii) the RNA translational enhancer of enceph alomyocarditis virus; and (iv) the genetic background of vaccinia viru s. In cells infected with the recombinant vaccinia virus, reporter bet a-galactosidase synthesis was not detected in the absence of inducer, An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor, Regulated synthesis of the secreted and highly glycosylated human immu nodeficiency virus 1 envelope protein gp120 was also demonstrated. Yie lds of both proteins were approximate to 2 mg per 108 cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or inc omplete open reading frames in recombinant vaccinia viruses are descri bed.