Application of Gal4/UAS System and MicroRNA Decoy Technology: Deciphering the Target of MicroRNA-275 in Aedes aegypti

Despite recent progress in genetic manipulations of mosquitoes, there is a much needed improvement and development of specific research tools to further facilitate research of these disease vectors. The focus of this dissertation is to extend and refine upon the Gal4/UAS system for the yellow fever mosquito, Aedes aegypti. The first part of the dissertation focuses on the development the blood-meal-activated, gut-specific Gal4/UAS system by utilizing a 1.1-kb, 5' upstream region of the carboxypeptidase A (CP) gene to genetically engineer the CP-Gal4 driver mosquito line. The generated driver successfully drive the expression of the effect gene followed a similar pattern as endogenous CP gene. Using the CP-Gal4/UAS-EGFP mosquitoes, we demonstrated that the CP gene is regulated by insulin and amino acids. In the second part of the dissertation, the gut-specificCP-Gal4/UAS and the fat-body specific Vg-Gal4/UAS systems were utilized in a combination with microRNA decoy to investigate the function of the, aae-miR-275 (miR-275). This approach allowed demonstrating a gut-specific function of miR-275.Specific miR-275 silencing in the midgut resulted in dramatic defects in blood digestion and egg development in female mosquitoes. The predicted miR-275 target gene- sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA)-was confirmed to directly bind miR-275 by means of luciferase assay. RNA interference silencing of SERCA or application of SERCA-specific inhibitor Thapsigargin (TG) resulted in an apparent lack of blood digestion, inhibition of egg development, as well as diminished egg number in comparison to controls. Application of the miR-275 mimic in the CP-Gal4/UAS-miR275decoy mosquitoes restored SERCA transcript level and rescued the phenotype. In the last part of this thesis, I described two additional midgut-specific Gal4 driver lines that are currently under development. Together, my work provide considerable advance in developing the binary Gal4/UAS system for research of essential gene and microRNA functions in the mosquito Ae. aegypti.