The recent development of mosquito genetic manipulation techniques and the completion of the Anopheles gambiae and Plasmodium falciparum genome sequencing projects opened up new scenarios both for basic research in mosquito biology and mosquito-parasite interaction and for the development of new vector control strategies. In this view the identification of tissue specific promoters is an essential tool for the proper employment of mosquito transformation technology. Stage and tissue-specific regulatory regions are highly desirable in order to control the expression of a chosen gene. Moreover, these characteristics are absolutely required when expressing anti-pathogen molecules in transgenic mosquitoes, both to minimize possible secondary effects and to improve efficiency by precise targeting. In this respect gut, salivary glands and haemocytes, because of their role in parasite transmission, are certainly among the most important targets. We report here the promoter analysis of the A. gambiae salivary gland genes AgApy and D7-related in the fruit fly Drosophila melanogaster and in the mosquito Anopheles stephensi. We initially analyzed AgApy, which encodes for the platelet inhibitor apyrase, and we could show that an 800 bp promoter fragment was able to drive a weak tissue-specific expression in the adult salivary glands of D. melanogaster (Lombardo F et al, 2000 J Biol Chem, 275: 23861-23868). In the meantime a technique for the genetic transformation of A. stephensi became available (Catteruccia F et al, 2000 Nature, 405: 959-962) and therefore we tested the same promoter fragment in this mosquito. The AgApy promoter-LacZ reporter-SV40 terminator cassette was transferred into a Minos-based vector marked with the GFP and used for transformation experiments. Six bona fide homozygous lines were established and characterized for the expression of the reporter gene by RT-PCR, histochemical staining and immunological means. RT-PCR analysis showed (i) LacZ transcription in adult males and females and (ii) expression of the transgene restricted to the salivary glands in two of the three lines analyzed. Histochemical staining revealed weak β-galactosidase activity in the female glands of individuals carrying multiple transgene insertions; in this case the staining was limited to the proximal portion of the medial lobe and to groups of cells in the lateral lobes. Finally, immuno-blot and immuno-fluorescence allowed to detect β-galactosidase only in the proximal portions of the salivary glands of transgenic females. This pattern differs from the endogenous expression profile of AgApy, which is restricted to the distal-lateral and medial lobes, suggesting that this promoter fragment may lack some tissue-specific enhancer or other essential regulatory elements. In our search for a strong salivary promoter we have also analyzed the regulatory regions of the A. gambiae D7r genes that are highly expressed in the female salivary glands (Arcà B et al, 2002 Insect Mol Biol, 11:47-55). The ~1 Kb regions located immediately upstream of the D7r2 and D7r4 genes were initially tested in D. melanogaster and, in a second step, the D7r4 promoter was analyzed by A. stephensi transformation. Histochemical analysis revealed intense β-galactosidase activity in the salivary glands of both adult male and female fruit flies. Inversely, RT-PCR analysis of transgenic mosquitoes transformed with the D7r4 promoter fragment showed only low LacZ transcription levels in adult males and females, with two out of five lines displaying an expression profile restricted to the salivary glands. The low expression level did not allow detection of β-galactosidase neither by Western blot, immuno-fluorescence or histochemical assay. The results obtained in the fruit fly suggest at least a partial conservation of “basic” salivary gland regulatory elements between the two species whereas the discrepancies that we observed between Drosophila and Anopheles may be connected to “specialized” additional control elements acquired by the mosquitoes during the evolution of haematophagy.

An Anopheles gambiae salivary gland promoter analysis in transgenic mosquitoes and fruit flies.

ARCA', BRUNO
2004

Abstract

The recent development of mosquito genetic manipulation techniques and the completion of the Anopheles gambiae and Plasmodium falciparum genome sequencing projects opened up new scenarios both for basic research in mosquito biology and mosquito-parasite interaction and for the development of new vector control strategies. In this view the identification of tissue specific promoters is an essential tool for the proper employment of mosquito transformation technology. Stage and tissue-specific regulatory regions are highly desirable in order to control the expression of a chosen gene. Moreover, these characteristics are absolutely required when expressing anti-pathogen molecules in transgenic mosquitoes, both to minimize possible secondary effects and to improve efficiency by precise targeting. In this respect gut, salivary glands and haemocytes, because of their role in parasite transmission, are certainly among the most important targets. We report here the promoter analysis of the A. gambiae salivary gland genes AgApy and D7-related in the fruit fly Drosophila melanogaster and in the mosquito Anopheles stephensi. We initially analyzed AgApy, which encodes for the platelet inhibitor apyrase, and we could show that an 800 bp promoter fragment was able to drive a weak tissue-specific expression in the adult salivary glands of D. melanogaster (Lombardo F et al, 2000 J Biol Chem, 275: 23861-23868). In the meantime a technique for the genetic transformation of A. stephensi became available (Catteruccia F et al, 2000 Nature, 405: 959-962) and therefore we tested the same promoter fragment in this mosquito. The AgApy promoter-LacZ reporter-SV40 terminator cassette was transferred into a Minos-based vector marked with the GFP and used for transformation experiments. Six bona fide homozygous lines were established and characterized for the expression of the reporter gene by RT-PCR, histochemical staining and immunological means. RT-PCR analysis showed (i) LacZ transcription in adult males and females and (ii) expression of the transgene restricted to the salivary glands in two of the three lines analyzed. Histochemical staining revealed weak β-galactosidase activity in the female glands of individuals carrying multiple transgene insertions; in this case the staining was limited to the proximal portion of the medial lobe and to groups of cells in the lateral lobes. Finally, immuno-blot and immuno-fluorescence allowed to detect β-galactosidase only in the proximal portions of the salivary glands of transgenic females. This pattern differs from the endogenous expression profile of AgApy, which is restricted to the distal-lateral and medial lobes, suggesting that this promoter fragment may lack some tissue-specific enhancer or other essential regulatory elements. In our search for a strong salivary promoter we have also analyzed the regulatory regions of the A. gambiae D7r genes that are highly expressed in the female salivary glands (Arcà B et al, 2002 Insect Mol Biol, 11:47-55). The ~1 Kb regions located immediately upstream of the D7r2 and D7r4 genes were initially tested in D. melanogaster and, in a second step, the D7r4 promoter was analyzed by A. stephensi transformation. Histochemical analysis revealed intense β-galactosidase activity in the salivary glands of both adult male and female fruit flies. Inversely, RT-PCR analysis of transgenic mosquitoes transformed with the D7r4 promoter fragment showed only low LacZ transcription levels in adult males and females, with two out of five lines displaying an expression profile restricted to the salivary glands. The low expression level did not allow detection of β-galactosidase neither by Western blot, immuno-fluorescence or histochemical assay. The results obtained in the fruit fly suggest at least a partial conservation of “basic” salivary gland regulatory elements between the two species whereas the discrepancies that we observed between Drosophila and Anopheles may be connected to “specialized” additional control elements acquired by the mosquitoes during the evolution of haematophagy.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11588/303052
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