High-efficiency protein synthesis from T7 RNA polymerase transcripts in 3T3 fibroblasts.
After NIH3T3 cells constitutively expressing T7 RNA polymerase were transfected (+ Ca.phosphate) with a circular DNA containing the firefly luciferase(Luc)-encoding gene (luc) 3' to the encephalomyocarditis (EMC) virus 5'-untranslated sequence and T7 promoter, Luc protein comprising approx. 20% of total cellular protein was obtained. After similar transfection of an analogous construct containing the lacZ gene into the same cell line, at least 50% of the cells produced beta-galactosidase. Fibroblasts lipofected with uncapped RNA transcripts containing EMC sequence expressed the reporter genes as efficiently as capped transcripts. A novel approach was used to generate RNA transcripts containing poly(A) at its very 3' end. RNA from a luc vector with a poly(A) sequence at the very 3' end produced 20-fold more Luc than the RNA from the same vector with an additional 3' nonpoly(A) sequence. These results suggest that this T7 RNA polymerase expression system will be useful for the efficient production of proteins in mammalian cells.
Deng
H
H
Department of Pediatrics, Waisman Center, University of Wisconsin, Madison 53705.
Wang
C
C
Acsadi
G
G
Wolff
J A
JA
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004270:DNA, Circular / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004686:Encephalomyocarditis virus / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D050356:Lipid Metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000235:genetics
D004270:DNA, Circular; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014764:Viral Proteins; D011061:Poly A; D008156:Luciferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase