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 cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi 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 beta-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 immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.
Ward
G A
GA
MedImmune, Inc., Gaithersburg, MD 20878, USA.
Stover
C K
CK
Moss
B
B
Fuerst
T R
TR
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis / Q000235:genetics; D006358:Hot Temperature; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase