A coumermycin/novobiocin-regulated gene expression system.
Regulated expression of transgenes in mammals is an important technique in both functional genomic studies and clinical applications. Here we describe a regulated gene expression system for mammals, based on coumarin-switched dimerization of the bacterial DNA gyrase B subunit (GyrB). The transactivator was constructed by fusing the GyrB activator to the bacterial lambda repressor-binding domain. The antibiotic coumermycin in nanomolar concentrations activated the transgene through binding of the homodimerized chimeric transactivator to the lambda operator located upstream of a minipromoter. More significantly, addition of novobiocin, an antagonist of coumermycin, promptly switched off expression of the gene by abolishing coumermycin-induced dimerization of the transactivator. Site-directed mutagenesis of the lambda repressor-binding domain resulted in significant reduction of basal expression levels and an induction reaching four orders of magnitude in stably transfected 293A cells in response to coumermycin. The capability of this inducible system for tightly regulated gene expression was demonstrated by the ready generation of stable cell lines inducibly expressing the proapoptotic bax gene in mammalian cells. Hence, this novel coumarin switch-on/switch-off system should broaden the utility of regulated gene expression, particularly when rapid on/off interchange is required.
Zhao
Hui-Fen
HF
Mammalian Cell Genetics, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada H4P 2R2.
Boyd
Jason
J
Jolicoeur
Normand
N
Shen
Shi-Hsiang
SH
D049933:Aminocoumarins; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002460:Cell Line; D003374:Coumarins / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics; D019281:Dimerization; D005434:Flow Cytometry; D015870:Gene Expression; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D020014:K562 Cells; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009675:Novobiocin / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D019076:Transgenes
D049933:Aminocoumarins; D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D003374:Coumarins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D009675:Novobiocin; D008156:Luciferases; D027081:DNA Gyrase; C004628:coumermycin