Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for basic research in mosquito biology and mosquito-parasite interaction and for the development of new vector control strategies. Recently, we described the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4 in transgenic An. stephensi. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic An. stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-lateral rather than in the distal-lateral lobes of female glands. We report here the establishment of transgenic An. gambiae lines carrying a larger portion of the AgApy promoter (~ 2,4 kb) driving the LacZ reporter gene. A transformation vector based on the piggyBac transposon and marked with the dsRed gene under control of the 3xP3 promoter was used for the microinjection experiments. A number of different transgenic An. gambiae G1 founders were obtained: the progeny was analyzed by Southern blot revealing the presence of single as well as multiple copies of the transgene. Three independent lines containing one (E9), three (D6) and four copies (D4) of the transgene were selected and established for further analysis. RT-PCR expression analysis with LacZ-specific primers showed different expression profiles in the three transgenic lines suggesting that position effect and copy number affect reporter gene expression. In fact, LacZ mRNA was ubiquitously detectable in transgenic mosquitoes from the D4 line whereas individuals from the D6 line exhibited an expression profile restricted to female salivary glands and adult males. Finally, transgene expression in E9 mosquitoes appeared strong in larval and pupal stages, absent in female carcasses and rather weak in salivary glands and males. Hystochemical assays on whole glands and western blot analysis on salivary protein extracts failed so far to reveal β-galactosidase protein in transgenic mosquitoes of the different lines.

Anopheles gambiae salivary gland promoter analysis.

ARCA', BRUNO
2005

Abstract

Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for basic research in mosquito biology and mosquito-parasite interaction and for the development of new vector control strategies. Recently, we described the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4 in transgenic An. stephensi. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic An. stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-lateral rather than in the distal-lateral lobes of female glands. We report here the establishment of transgenic An. gambiae lines carrying a larger portion of the AgApy promoter (~ 2,4 kb) driving the LacZ reporter gene. A transformation vector based on the piggyBac transposon and marked with the dsRed gene under control of the 3xP3 promoter was used for the microinjection experiments. A number of different transgenic An. gambiae G1 founders were obtained: the progeny was analyzed by Southern blot revealing the presence of single as well as multiple copies of the transgene. Three independent lines containing one (E9), three (D6) and four copies (D4) of the transgene were selected and established for further analysis. RT-PCR expression analysis with LacZ-specific primers showed different expression profiles in the three transgenic lines suggesting that position effect and copy number affect reporter gene expression. In fact, LacZ mRNA was ubiquitously detectable in transgenic mosquitoes from the D4 line whereas individuals from the D6 line exhibited an expression profile restricted to female salivary glands and adult males. Finally, transgene expression in E9 mosquitoes appeared strong in larval and pupal stages, absent in female carcasses and rather weak in salivary glands and males. Hystochemical assays on whole glands and western blot analysis on salivary protein extracts failed so far to reveal β-galactosidase protein in transgenic mosquitoes of the different lines.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/303082
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