Stringent Chemical and Thermal Regulation of Recombinant Gene Expression by Vaccinia Virus Vectors in Mammalian Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 15; pp. 6773 - 6777
• Main Authors: Ward, George A., Stover, C. Kendall, Moss, Bernard, Fuerst, Thomas R.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 18.07.1995; National Acad Sciences; National Academy of Sciences
• Subjects: AIDS/HIV; Animals; Bacteria; Base Sequence; beta-Galactosidase - biosynthesis; beta-Galactosidase - genetics; Cells, Cultured; Cloning, Molecular - methods; encephalomyocarditis virus; Enzyme Induction; Escherichia coli; Gene Expression Regulation; Genes; Genetic vectors; Genetic Vectors - genetics; Genetics; HIV 1; HIV Envelope Protein gp120 - biosynthesis; HIV Envelope Protein gp120 - genetics; Hot Temperature; human immunodeficiency virus 1; Isopropyl Thiogalactoside - pharmacology; Mammals; Mathematical vectors; Molecular Sequence Data; phage T7; Recombinant Proteins - biosynthesis; Regulatory Sequences, Nucleic Acid - genetics; Repressor proteins; Ribonucleic acid; RNA; Vaccinia; Vaccinia virus; Vaccinia virus - genetics; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.15.6773

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, φ10 promoter, and Tφ transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter β-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 β-D-thiogalactopyranoside or by temperature elevation from 30 to 37⚬C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were ≈2 mg per 108cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.