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Genetics, Vol. 167, 1781-1790, August 2004, Copyright © 2004
doi:10.1534/genetics.104.028175
Conditional Expression in the Malaria Mosquito Anopheles stephensi With Tet-On and Tet-Off Systems
Gareth J. Lycett*,
Fotis C. Kafatos* and
Thanasis G. Loukeris*,
,1
* European Molecular Biology Laboratory, Heidelberg D-69117, Germany
Institute of Molecular Biology and Biotechnology, FORTH, GR71110 Heraklion, Crete, Greece
1 Corresponding author: Institute of Molecular Biology and Biotechnology, FORTH, Vassilika Vouton, P.O. Box 1527, GR 711 10 Heraklion, Crete, Greece.
E-mail: loukeris{at}imbb.forth.gr
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 ß-galactosidase under dual, tissue-specific and dox-mediated regulation. In adult rtTA/TetOPlacZ progeny, dox treatment rapidly induced ß-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.
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