Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes.
Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc. Natl. Acad. Sci. USA. 86, 6126-6130, 1989). We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes. Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes. The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter. The system was optimized in terms of enzyme, DNA and liposome concentrations. Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells. The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells. The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene. We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.
Gao
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Department of Pharmacology, University of Pittsburgh School of Medicine, PA 15261.
Huang
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D017123:Bacteriophage T7 / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008175:Lung Neoplasms; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
D008081:Liposomes; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases