Synthesis of functional mRNA in mammalian cells by bacteriophage T3 RNA polymerase.
We found that the 5' nontranslated leader sequence from encephalomyocarditis virus (EMCV) allowed transcripts that were synthesized by the T3 RNA polymerase in mammalian cells to be translated in a cap-independent fashion. Stable mouse cell lines that carry the T3 RNA polymerase gene expressed the chloramphenicol acetyltransferase (CAT) gene under the control of a phage promoter when the CAT gene was fused to the EMCV leader and introduced into the cells by transient DNA uptake. The level of gene expression in such cells was similar to or greater than that observed with a conventional transient expression vector that is dependent on transcription by the host RNA polymerase II. Expression of the EMCV-CAT fusion gene was stimulated by cotransfection of the cells with a gene that encodes the poliovirus protease 2A protein (which inhibits cap-dependent translation), demonstrating that the EMCV-CAT fusion gene was expressed in a cap-independent fashion. Introduction of both the T3 RNA polymerase gene and the EMCV-CAT fusion gene into a variety of cultured mammalian cell lines (HeLa, BSC40, Ltk-, NIH 3T3, and C127) demonstrated that the T3-EMCV expression system functions in a broad range of cell types.
Zhou
Y
Y
Department of Microbiology and Immunology, Morse Institute of Molecular Genetics, State University of New York, Health Science Center, Brooklyn 11203-2098.
Giordano
T J
TJ
Durbin
R K
RK
McAllister
W T
WT
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004686:Encephalomyocarditis virus / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D015678:Viral Structural Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases