Gene Silencing via dsRNA Microinjection in Sand Flies

Gene-specific primers (dsAtt-F, dsAtt-R, dsDef2-F, and dsDef2-R) coupled to a T7 promoter sequence (Table S1) were designed to amplify templates from sand fly cDNA by PCR. dsLacZ-F and dsLacZ-R primers were used to amplify the template from p-GEM-T Easy plasmid (Promega) as control dsRNA. Touchdown PCR was used as follows: 95 °C for 3 min; 16 cycles of 95 °C for 45 s, 68 to 50 °C (progressively decreasing 1 °C per cycle) for 45 s, and 72 °C for 45 s; 26 cycles of 95 °C for 45 s, 50 °C for 45 s, and 72 °C for 45 s; 72 °C for 3 min. These templates were purified by Wizard SV Gel and PCR cleanup system (Promega) and used in dsRNA synthesis reaction by MEGAscript RNAi kit (Invitrogen) following the manufacturer’s instructions. The produced dsRNA was lyophilized and resuspended in ultrapure H₂O to 4.5 μg/μL final concentration. Sand flies were microinjected intrathoracically with 32.2 nL of dsRNA using Nanoject II microinjector (Drummond) [39 (link)].