Analysis of apyrase 5' upstream region validates improved Anopheles gambiae transformation technique

We have analyzed the upstream regulatory sequence of the An. gambiae salivary gland-specific apyrase (AgApy) gene in transgenic An. gambiae using a piggyBac transposable element vector marked by a 3xP3 promoter:DsRed gene fusion. Efficient germ-line transformation in An. gambiae mosquitoes was obtained and several integration events in at least three different G0 families were detected. LacZ reporter gene expression was analyzed in three transgenic lines/groups, and in only one group was tissue-specific expression restricted to salivary glands.Our data describe an efficient genetic transformation of An. gambiae embryos. However, expression from the selected region of the AgApy promoter is weak and position effects may mask tissue- and stage- specific activity in transgenic mosquitoes.The mosquito Anopheles gambiae is the main vector of the human malaria parasite Plasmodium falciparum in sub-Saharan Africa. Within the insect, the parasite undergoes a complex life-cycle that includes fertilization, midgut invasion, sporozoite maturation, avoidance of the mosquito innate immune response and, as prerequisite for a successful transmission, recognition and entrance into the salivary glands [1]. The development of tools for mosquito genetic manipulation have provided evidence that Plasmodium development can be modified in the anopheline vector and opened new perspectives for studies on vector biology and on parasite-vector-host interactions [2,3].Several studies in the last decade reported the successful use of tissue-specific promoters for directing the expression of exogenous genes in different mosquito target organs (primarily midgut, hemocoel and salivary glands), mainly in the yellow fever vector Aedes aegypti and in the Asian malaria vector Anopheles stephensi [4-7]. As far as the main African malaria vector An. gambiae is concerned, after the initial successful transformation [8] only one additional study with transgenic An. gambiae has been reported so far [9]. In