title: Conditional expression in the malaria mosquito Anopheles stephensi with Tet-On and Tet-Off systems.
abstract: We report successful conditional gene expression in the malaria vector, Anopheles stephensi, on the basis of binary systems consisting of gene driver and responder transgenic lines generated by Minos-mediated germline transformation. An A. gambiae tissue-specific enhancer derived from a serpin (SRPN10) gene was utilized to control the temporal and spatial expression of doxycycline (dox)-sensitive transcriptional regulators in the driver lines. The "Tet-Off" driver utilized the tetracycline-controlled transcriptional activator (tTA) that is unable to bind and activate transcription from tetracycline operators (TetO) in the presence of dox; the "Tet-on" driver utilized the reverse tTA (rtTA) that, conversely, binds and activates TetO operators in the presence of dox. The responder lines carried insertions encompassing a LacZ reporter gene, cis-regulated by a TetO-P-element hybrid promoter. The progeny of crosses between driver and responder lines expressed beta-galactosidase under dual, tissue-specific and dox-mediated regulation. In adult rtTA/TetOPlacZ progeny, dox treatment rapidly induced beta-galactosidase activity throughout the midgut epithelium and especially in malaria parasite-invaded epithelial cells. Transactivator-dependent, dox-mediated regulation was observed in hemocytes and pericardial cells using both systems. Conditional tissue-specific regulation is a powerful tool for analyzing gene function in mosquitoes and potentially for development of strategies to control disease transmission.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg D-69117, Germany.
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Lycett
authors.forename: Fotis C
authors.initials: FC
authors.lastname: Kafatos
authors.forename: Thanasis G
authors.initials: TG
authors.lastname: Loukeris
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000852:Anopheles / Q000235:genetics*; D004318:Doxycycline / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008288:Malaria / Q000635:transmission; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D004318:Doxycycline