Skip to main content
Genetics logoLink to Genetics
. 2004 Aug;167(4):1781–1790. doi: 10.1534/genetics.104.028175

Conditional expression in the malaria mosquito Anopheles stephensi with Tet-On and Tet-Off systems.

Gareth J Lycett 1, Fotis C Kafatos 1, Thanasis G Loukeris 1
PMCID: PMC1471022  PMID: 15342516

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.

Full Text

The Full Text of this article is available as a PDF (389.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bello B., Resendez-Perez D., Gehring W. J. Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system. Development. 1998 Jun;125(12):2193–2202. doi: 10.1242/dev.125.12.2193. [DOI] [PubMed] [Google Scholar]
  2. Benedict Mark Q., Robinson Alan S. The first releases of transgenic mosquitoes: an argument for the sterile insect technique. Trends Parasitol. 2003 Aug;19(8):349–355. doi: 10.1016/s1471-4922(03)00144-2. [DOI] [PubMed] [Google Scholar]
  3. Berens Christian, Hillen Wolfgang. Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. Eur J Biochem. 2003 Aug;270(15):3109–3121. doi: 10.1046/j.1432-1033.2003.03694.x. [DOI] [PubMed] [Google Scholar]
  4. Blandin Stephanie, Shiao Shin-Hong, Moita Luis F., Janse Chris J., Waters Andrew P., Kafatos Fotis C., Levashina Elena A. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell. 2004 Mar 5;116(5):661–670. doi: 10.1016/s0092-8674(04)00173-4. [DOI] [PubMed] [Google Scholar]
  5. Blandin Stéphanie, Moita Luis F., Köcher Thomas, Wilm Matthias, Kafatos Fotis C., Levashina Elena A. Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene. EMBO Rep. 2002 Aug 16;3(9):852–856. doi: 10.1093/embo-reports/kvf180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brand A. H., Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  7. Brodu V., Mugat B., Roignant J. Y., Lepesant J. A., Antoniewski C. Dual requirement for the EcR/USP nuclear receptor and the dGATAb factor in an ecdysone response in Drosophila melanogaster. Mol Cell Biol. 1999 Aug;19(8):5732–5742. doi: 10.1128/mcb.19.8.5732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Catteruccia F., Nolan T., Loukeris T. G., Blass C., Savakis C., Kafatos F. C., Crisanti A. Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature. 2000 Jun 22;405(6789):959–962. doi: 10.1038/35016096. [DOI] [PubMed] [Google Scholar]
  9. Christophides George K., Zdobnov Evgeny, Barillas-Mury Carolina, Birney Ewan, Blandin Stephanie, Blass Claudia, Brey Paul T., Collins Frank H., Danielli Alberto, Dimopoulos George. Immunity-related genes and gene families in Anopheles gambiae. Science. 2002 Oct 4;298(5591):159–165. doi: 10.1126/science.1077136. [DOI] [PubMed] [Google Scholar]
  10. Danielli A., Loukeris T. G., Lagueux M., Müller H. M., Richman A., Kafatos F. C. A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles gambiae. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7136–7141. doi: 10.1073/pnas.97.13.7136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Danielli Alberto, Kafatos Fotis C., Loukeris Thanasis G. Cloning and characterization of four Anopheles gambiae serpin isoforms, differentially induced in the midgut by Plasmodium berghei invasion. J Biol Chem. 2002 Nov 26;278(6):4184–4193. doi: 10.1074/jbc.M208187200. [DOI] [PubMed] [Google Scholar]
  12. Dimopoulos George, Christophides George K., Meister Stephan, Schultz Jörg, White Kevin P., Barillas-Mury Carolina, Kafatos Fotis C. Genome expression analysis of Anopheles gambiae: responses to injury, bacterial challenge, and malaria infection. Proc Natl Acad Sci U S A. 2002 Jun 19;99(13):8814–8819. doi: 10.1073/pnas.092274999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gardner Malcolm J., Hall Neil, Fung Eula, White Owen, Berriman Matthew, Hyman Richard W., Carlton Jane M., Pain Arnab, Nelson Karen E., Bowman Sharen. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002 Oct 3;419(6906):498–511. doi: 10.1038/nature01097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gill G., Ptashne M. Negative effect of the transcriptional activator GAL4. Nature. 1988 Aug 25;334(6184):721–724. doi: 10.1038/334721a0. [DOI] [PubMed] [Google Scholar]
  15. Gossen Manfred, Bujard Hermann. Studying gene function in eukaryotes by conditional gene inactivation. Annu Rev Genet. 2002 Jun 11;36:153–173. doi: 10.1146/annurev.genet.36.041002.120114. [DOI] [PubMed] [Google Scholar]
  16. Grossman G. L., Rafferty C. S., Clayton J. R., Stevens T. K., Mukabayire O., Benedict M. Q. Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element. Insect Mol Biol. 2001 Dec;10(6):597–604. doi: 10.1046/j.0962-1075.2001.00299.x. [DOI] [PubMed] [Google Scholar]
  17. Han Y. S., Thompson J., Kafatos F. C., Barillas-Mury C. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J. 2000 Nov 15;19(22):6030–6040. doi: 10.1093/emboj/19.22.6030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hoffmann A., Villalba M., Journot L., Spengler D. A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines. Nucleic Acids Res. 1997 Mar 1;25(5):1078–1079. doi: 10.1093/nar/25.5.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Holt Robert A., Subramanian G. Mani, Halpern Aaron, Sutton Granger G., Charlab Rosane, Nusskern Deborah R., Wincker Patrick, Clark Andrew G., Ribeiro José M. C., Wides Ron. The genome sequence of the malaria mosquito Anopheles gambiae. Science. 2002 Oct 4;298(5591):129–149. doi: 10.1126/science.1076181. [DOI] [PubMed] [Google Scholar]
  20. Horn Carsten, Wimmer Ernst A. A transgene-based, embryo-specific lethality system for insect pest management. Nat Biotechnol. 2002 Dec 16;21(1):64–70. doi: 10.1038/nbt769. [DOI] [PubMed] [Google Scholar]
  21. Kokoza V. A., Martin D., Mienaltowski M. J., Ahmed A., Morton C. M., Raikhel A. S. Transcriptional regulation of the mosquito vitellogenin gene via a blood meal-triggered cascade. Gene. 2001 Aug 22;274(1-2):47–65. doi: 10.1016/s0378-1119(01)00602-3. [DOI] [PubMed] [Google Scholar]
  22. Krafsur E. S., Townson H., Davidson G., Curtis C. F. Screwworm eradication is what it seems. Nature. 1986 Oct 9;323(6088):495–496. doi: 10.1038/323495b0. [DOI] [PubMed] [Google Scholar]
  23. Krebs R. A., Feder M. E. Tissue-specific variation in Hsp70 expression and thermal damage in Drosophila melanogaster larvae. J Exp Biol. 1997 Jul;200(Pt 14):2007–2015. doi: 10.1242/jeb.200.14.2007. [DOI] [PubMed] [Google Scholar]
  24. Levashina E. A., Moita L. F., Blandin S., Vriend G., Lagueux M., Kafatos F. C. Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae. Cell. 2001 Mar 9;104(5):709–718. doi: 10.1016/s0092-8674(01)00267-7. [DOI] [PubMed] [Google Scholar]
  25. Lis J. T., Simon J. A., Sutton C. A. New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene. Cell. 1983 Dec;35(2 Pt 1):403–410. doi: 10.1016/0092-8674(83)90173-3. [DOI] [PubMed] [Google Scholar]
  26. Meister Marie, Lagueux Marie. Drosophila blood cells. Cell Microbiol. 2003 Sep;5(9):573–580. doi: 10.1046/j.1462-5822.2003.00302.x. [DOI] [PubMed] [Google Scholar]
  27. Merika M., Orkin S. H. Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Krüppel family proteins Sp1 and EKLF. Mol Cell Biol. 1995 May;15(5):2437–2447. doi: 10.1128/mcb.15.5.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mismer D., Rubin G. M. Analysis of the promoter of the ninaE opsin gene in Drosophila melanogaster. Genetics. 1987 Aug;116(4):565–578. doi: 10.1093/genetics/116.4.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Müller H. M., Dimopoulos G., Blass C., Kafatos F. C. A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes. J Biol Chem. 1999 Apr 23;274(17):11727–11735. doi: 10.1074/jbc.274.17.11727. [DOI] [PubMed] [Google Scholar]
  30. Osta Mike A., Christophides George K., Kafatos Fotis C. Effects of mosquito genes on Plasmodium development. Science. 2004 Mar 26;303(5666):2030–2032. doi: 10.1126/science.1091789. [DOI] [PubMed] [Google Scholar]
  31. Smith R. L., Geller A. I., Escudero K. W., Wilcox C. L. Long-term expression in sensory neurons in tissue culture from herpes simplex virus type 1 (HSV-1) promoters in an HSV-1-derived vector. J Virol. 1995 Aug;69(8):4593–4599. doi: 10.1128/jvi.69.8.4593-4599.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thomas D. D., Donnelly C. A., Wood R. J., Alphey L. S. Insect population control using a dominant, repressible, lethal genetic system. Science. 2000 Mar 31;287(5462):2474–2476. doi: 10.1126/science.287.5462.2474. [DOI] [PubMed] [Google Scholar]
  33. Urlinger S., Baron U., Thellmann M., Hasan M. T., Bujard H., Hillen W. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7963–7968. doi: 10.1073/pnas.130192197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Watanabe K., Clarke T. R., Lane A. H., Wang X., Donahoe P. K. Endogenous expression of Müllerian inhibiting substance in early postnatal rat sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites. Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1624–1629. doi: 10.1073/pnas.97.4.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zdobnov Evgeny M., von Mering Christian, Letunic Ivica, Torrents David, Suyama Mikita, Copley Richard R., Christophides George K., Thomasova Dana, Holt Robert A., Subramanian G. Mani. Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science. 2002 Oct 4;298(5591):149–159. doi: 10.1126/science.1077061. [DOI] [PubMed] [Google Scholar]
  36. Zhuang Z., Linser P. J., Harvey W. R. Antibody to H(+) V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedes aegypti). J Exp Biol. 1999 Sep;202(Pt 18):2449–2460. doi: 10.1242/jeb.202.18.2449. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES