A Coumermycin/Novobiocin-regulated gene expression system

  1. Get@NRC: A Coumermycin/Novobiocin-regulated gene expression system (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1089/104303403322542266
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Journal titleHuman Gene Therapy
Pages16191629; # of pages: 11
SubjectDimerization; Gene Expression; genome; pha
AbstractRegulated 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 λ repressor-binding domain. The antibiotic coumermycin in nanomolar concentrations activated the transgene through binding of the homodimerized chimeric transactivator to the λ 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 λ 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.
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AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedNo
NRC number46163
NPARC number3538928
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Record identifiercc3840eb-759e-41c5-8157-3a2d9c9b09a7
Record created2009-03-01
Record modified2016-05-09
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