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

phiC31 integrase is a phage-derived recombinase enzyme that catalyzes the site-specific recombination of a transgene, a foreign gene, into a host genome. For transgene integration in an Anopheles host, begin with an Anopheles embryo in a suitable buffer.

The Anopheles genome is pre-engineered with an attachment site for gene integration and a cyan-fluorescent marker for visual selection. Next, add a small volume of donor plasmid carrying the desired transgene cargo, an attachment site, a red fluorescent marker, and plasmid backbone components to a vial. Then, add an optimum volume of a helper plasmid carrying a gene-encoding integrase. Now, load the plasmid mixture into a microsyringe.

Gently inject the plasmids into the embryo's posterior pole, the site of origin of germ cells, resulting in a slight movement of the cytoplasm. Once inside the embryo, the helper plasmid starts synthesizing integrase enzymes.

The integrase enzyme mediates site-specific recombination between the donor plasmid and the host genome. Thus, the transgene and the red fluorescent marker from the donor plasmid integrate within the recombinant attachment sites in the Anopheles genome. Thereafter, incubate the injected embryo under physiological conditions for hatching, with intermittent swirling.

Within days, the embryo hatches into a larva. Under a fluorescence microscope, the Anopheles larva with successfully integrated genes expresses both cyan fluorescence, marking the host genome, and red fluorescence, marking the transgene from the donor plasmid.

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.