title: Optimization of the Tet-On system for inducible expression of RAGE.
abstract: We have optimized a two-plasmid Tet-On system, the regulatory plasmid and the response plasmid, to produce tightly controlled inducible expression of the gene RAGE in cell-culture models. Two sets of plasmids were constructed: set 1 (universal; for broad range of cell types) and set 2 (neuron specific). For the response plasmid, the gene RAGE was cloned in pIRES2-EGFP plasmid (Clontech) and the CMV promoter replaced with TREtight (modified seven copies of Tet-operon fused with CMVm promoter). For the regulatory plasmid, rtTA (reverse tetracycline transactivator) was placed under either the CMV promoter or the cell-specific promoter neuronal specific enolase. Both plasmids have the mammalian selection marker neomycine; the EGFP reporter gene is only in the response plasmid and IRES is between the gene and EGFP. Following induction with doxycycline, cells expressing RAGE showed neomycine resistance and green fluorescence (EGFP). Our system has been tested in two different cell lines and showed negligible basal leakiness, high induction of the gene RAGE (142-fold), dose-dependent response to doxycycline, and strict cell-type specificity. This system is highly suitable for cell-specific expression of any gene of interest in primary cultures and mixed cell populations.
authors.affiliation: Department of Anatomy with Radiology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Shamim
authors.initials: S
authors.lastname: Shaikh
authors.forename: Louise F B
authors.initials: LF
authors.lastname: Nicholson
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018929:Cell Culture Techniques; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009355:Neomycin / Q000494:pharmacology; D009447:Neuroblastoma / Q000473:pathology; D052248:Nitric Oxide Synthase Type I / Q000235:genetics; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection
chemical_list: D000900:Anti-Bacterial Agents; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I; D013752:Tetracycline; D009355:Neomycin; D004318:Doxycycline