Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.
A mouse cell line that constitutively synthesizes the bacteriophage T7 RNA polymerase was constructed. Fluorescence microscopy indicated that the T7 RNA polymerase was present in the cytoplasmic compartment. The system provided, therefore, a unique opportunity to study structural elements of mRNA that affect stability and translation. The in vivo activity of the bacteriophage polymerase was demonstrated by transfection of a plasmid containing the chloramphenicol acetyltransferase (CAT) gene flanked by T7 promoter and termination signals. Synthesis of CAT was dependent on the presence of a cDNA copy of the untranslated region of encephalomyocarditis virus (ECMV) RNA downstream of the T7 promoter, consistent with the absence of RNA-capping activity in the cytoplasm. CAT expression from a plasmid, pT7EMCAT, containing the T7 and EMCV regulatory elements was detected within 4 hr after transfection and increased during the next 20 hr, exceeding that obtained by transfection of a plasmid with the CAT gene attached to a retrovirus promoter and enhancer. Nevertheless, the presumably cap-independent transient expression of CAT from pT7EMCAT was increased more than 500-fold when the transfected cells also were infected with wild-type vaccinia virus. A protocol for high-level expression involved the infection of the T7 RNA polymerase cell line with a single recombinant vaccinia virus containing the target gene regulated by a T7 promoter and EMCV untranslated region.
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
O
O
Elroy-Stein
B
B
Moss
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors; D051379:Mice; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics
D015678:Viral Structural Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases