Mosquito Embryo Microinjection: A Technique to Deliver Exogenous DNA into Embryo Yolk of Anopheles Gambiae to Generate Germline Mutations

To microinject exogenous DNA into the Anopheles embryo, take the desired number of freshly harvested, pre-blastoderm, mosquito eggs. At this early developmental stage, the egg contains nuclei without forming cellular structures.

Place the boat-shaped eggs at the periphery of a moist blotting paper present on the surface of a glass slide. This step ensures adequate hydration of the embryo for the ease of injection in the subsequent steps. Align the eggs adjacent to each other with their ventral surface exposed outward.

Now, take a narrow-bore microinjection needle containing the desired plasmid solution. Use a dissecting microscope to position the needle near the egg's posterior pole - a site for the germ cell precursors. Insert the needle at an appropriate angle that it pierces the exochorion - the outer layer - and the endochorion - an inner layer of the eggshell - eventually reaching the embryo.

Inject the plasmid mix into the yolk of the embryo. Gently retract the needle and allow the eggs to grow under suitable conditions. As the egg grows, some nuclei migrate to the posterior pole, where they can incorporate the microinjected DNA. These nuclei later become gametes - the sex cells that can result in heritable germline mutations.

Visualize the adult mosquitos for transformed phenotype - the observable characteristics in subsequent generations confirming successful microinjection.