phiC31-Integrase-Mediated Site-Directed Transgene Integration: A Microinjection Technique for Site-Specific Transgene Integration into an Anopheles Vector

Purify donor and helper plasmids using an endotoxin-free purification kit. Combine the attB-tagged donor plasmid carrying the transgene of interest, and the helper plasmid carrying the integrase, to obtain a mix with a final concentration of 350 nanograms per microliter of the donor plasmid, and 150 nanograms per microliter of the helper plasmid.

Precipitate the DNA by adding 0.1 volume of 3 molar sodium acetate, in 2.5 volumes of ice-cold 100% ethanol. Then, vortex. A white precipitate should be immediately visible. Wash the pellet, and resuspend it in injection buffer, to reach a total final concentration of 500 nanograms per microliter. Then, prepare aliquots of 10 to 15 microliters each, and store them at negative 20 degrees Celsius.

Blood feed 4 to 7-day-old mosquitoes from the desired docking line, and their wild-type counterparts, 72 hours prior to microinjection. Perform Anopheles gambiae embryo microinjections, in 25 millimolar sodium chloride, by targeting the posterior pole of the embryo at a 45-degree angle.

Perform Anopheles stephensi embryo microinjections, in halocarbon oil, by targeting the posterior pole at a 30-degree angle. Immediately after injection, transfer the eggs to a petri dish filled with sterile distilled water, and return them to insectary conditions. Upon hatching, transfer G₀ larvae into a tray with salted distilled water daily, and rear to pupae.