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.