title: Tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system.
abstract: The use of insect cells has been highly successful for the expression of foreign proteins from baculoviruses or plasmid vectors. Here, we describe a tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system. The system includes the DEF domains of the spruce budworm (Choristoneura fumiferana) EcR (CfEcR) fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and the EF domains of mammalian Mus musculus retinoid X receptor (MmRXR) fused to the acidic activation domains (AADs) of the baculovirus transactivators IE1 and IE0. Using a GAL4 response element in reporter constructs, both transient and stable expression in insect lepidopteran cells showed that the chimeric MmRXR and CfEcR only activated the reporter genes in the presence of inducer; no gene expression was detectable in the absence of inducer. Characterization of heterogenous activation domains in insect cells showed that the AADs from Autographa californica multiple nucleopolyhedrovirus (MNPV) IE1 and Orgyia pseudotsugata MNPV IE0 consistently exhibited higher inducible levels than the archetype AAD from herpesvirus VP16 in insect cells. To confirm the tight regulation of this system the highly toxic protein, diphtheria toxin (DT), was used. In the absence of an inducer no cytotoxic effect was observed in insect cells that had been transiently transformed with DT expressing plasmids. This system will therefore be a very useful tool for biotechnology applications expressing highly toxic proteins in insect cells and for studying the functional genomics of insects and microorganisms that infect them.
authors.affiliation: Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada V0H 1Z0.
authors.forename: Xiaojiang
authors.initials: X
authors.lastname: Dai
authors.forename: Leslie G
authors.initials: LG
authors.lastname: Willis
authors.forename: Subba R
authors.initials: SR
authors.lastname: Palli
authors.forename: David A
authors.initials: DA
authors.lastname: Theilmann
mesh_terms: D000818:Animals; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D018014:Gene Transfer Techniques; D007814:Larva; D007915:Lepidoptera; D051379:Mice; D010957:Plasmids; D011987:Receptors, Steroid / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology
chemical_list: D004167:Diphtheria Toxin; D011987:Receptors, Steroid; C055469:ecdysone receptor