High level gene expression in mammalian cells by a nuclear T7-phase RNA polymerase.
Here we describe a novel expression system for mammalian cells which is based on transcription of hybrid genes containing T7 phage promoters by a T7 phage RNA polymerase targeted to the nucleus of the host cells. The RNA polymerase gene of T7 phage has been modified by substituting a sequence encoding the nuclear location signal of SV40 large T antigen for the N-terminal part of the polymerase gene. Expression of the modified gene is driven by the mouse metallothionein promoter in transfected mouse Ltk- cells resulting in high concentration of the polymerase in the nucleus. Nuclear T7 RNA polymerase directs efficient transcription of the cat gene under control of a T7 promoter. T7 constructs are expressed at a level at least 6 fold higher than the prototype pRSVcat. The unique properties of this heterologeous expression system are discussed.
Lieber
A
A
Akademie der Wissenschaften der DDR, Zentralinstitut für Molekularbiologie, Berlin-Buch, GDR.
Kiessling
U
U
Strauss
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D008322:Mammals; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000201:enzymology*; D014158:Transcription, Genetic
D000952:Antigens, Polyomavirus Transforming; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases