The availability of stage and tissue-specific promoters represents an essential tool to fully exploit the recently developed mosquito transformation technology. In particular, regulatory regions driving expression into the gut, salivary glands and haemocytes of adult females are highly desirable considering the role of these organs in parasite transmission. Moreover, a tight control of transgene expression would also be important to minimize possible secondary effects and to improve efficiency by precise targeting. With the objective to identify Anopheles gambiae salivary gland promoters we focused our attention on the salivary genes AgApy, which encodes the platelet inhibitor apyrase, and D7-related. An 800 bp promoter fragment from the AgApy gene, previously shown to drive a weak but tissue-specific expression in adult Drosophila melanogaster salivary glands, was analyzed in the mosquito Anopheles stephensi. RT-PCR transcriptional analysis of transgenic A. stephensi mosquitoes revealed expression of the LacZ reporter gene both in adult males and females and specifically in the salivary glands of two of the three lines analyzed. However, weak β-galactosidase activity was detectable only in the salivary glands of individuals carrying multiple transgene insertions. Finally, immuno-blot and immuno-fluorescence indicated that transgene expression was restricted to the proximal portions of females salivary glands. This pattern differs from the endogenous expression profile of AgApy, which is restricted to the distal-lateral and medial lobes, suggesting that the promoter fragment used may lack some essential regulatory elements. We also analyzed the putative promoters of the highly expressed A. gambiae D7r genes. Both the D7r2 and D7r4 promoters worked nicely in the fruit fly where intense β-galactosidase activity was revealed in the adult salivary glands of both sexes. Surprisingly, only low LacZ transcription levels (with gland-specificity in two out of five lines) and no β-galactosidase activity were found in transgenic mosquitoes carrying the D7r4 promoter. Overall the fruit fly analysis suggests a partial conservation of some “basic” salivary gland regulatory elements between the two species, whereas the discrepancies observed between Drosophila and Anopheles could be explained by a “specialized” additional control elements perhaps acquired by the mosquitoes during the evolution of haematophagy.

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

LOMBARDO, Fabrizio;LANFRANCOTTI, Alessandra;COLUZZI BARTOCCIONI, Caio Mario;ARCA', Bruno
2004

Abstract

The availability of stage and tissue-specific promoters represents an essential tool to fully exploit the recently developed mosquito transformation technology. In particular, regulatory regions driving expression into the gut, salivary glands and haemocytes of adult females are highly desirable considering the role of these organs in parasite transmission. Moreover, a tight control of transgene expression would also be important to minimize possible secondary effects and to improve efficiency by precise targeting. With the objective to identify Anopheles gambiae salivary gland promoters we focused our attention on the salivary genes AgApy, which encodes the platelet inhibitor apyrase, and D7-related. An 800 bp promoter fragment from the AgApy gene, previously shown to drive a weak but tissue-specific expression in adult Drosophila melanogaster salivary glands, was analyzed in the mosquito Anopheles stephensi. RT-PCR transcriptional analysis of transgenic A. stephensi mosquitoes revealed expression of the LacZ reporter gene both in adult males and females and specifically in the salivary glands of two of the three lines analyzed. However, weak β-galactosidase activity was detectable only in the salivary glands of individuals carrying multiple transgene insertions. Finally, immuno-blot and immuno-fluorescence indicated that transgene expression was restricted to the proximal portions of females salivary glands. This pattern differs from the endogenous expression profile of AgApy, which is restricted to the distal-lateral and medial lobes, suggesting that the promoter fragment used may lack some essential regulatory elements. We also analyzed the putative promoters of the highly expressed A. gambiae D7r genes. Both the D7r2 and D7r4 promoters worked nicely in the fruit fly where intense β-galactosidase activity was revealed in the adult salivary glands of both sexes. Surprisingly, only low LacZ transcription levels (with gland-specificity in two out of five lines) and no β-galactosidase activity were found in transgenic mosquitoes carrying the D7r4 promoter. Overall the fruit fly analysis suggests a partial conservation of some “basic” salivary gland regulatory elements between the two species, whereas the discrepancies observed between Drosophila and Anopheles could be explained by a “specialized” additional control elements perhaps 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: https://hdl.handle.net/11573/472839
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