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

JoVE Encyclopedia of Experiments

Biologische Technieken

0 weergaven • 3:12 min • July 8th, 2025

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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.

Use a micro-loader tip to fill the needles with two microliters of DNA mixture. Insert the needle into the needle-holder, and connect the automated pressure pump tubing. Align the needle so that it makes an angle of 15 degrees with the slide's plane.

Open the needle tip by carefully touching the first egg. Then, insert the needle’s tip to about 10 micrometers in the posterior pole. A successful injection will lead to a small movement of the cytoplasm within the egg.

Use the microscope co-axial stage controls to move to the next egg to continue injection. To ensure that the needle tip remains open and has not been clogged, press the 'Inject' button before entering another embryo, and visualize the small droplet at the opening of the needle. If the needle gets clogged, press the 'Clear' button to clear the needle, and repeat the droplet visualization test.

Adjust the pressure as needed, if the needle tip opening gets slightly bigger to ensure that the droplet size stays small. Inject about 40 to 50 eggs with one needle. After injections are complete, rinse the eggs off into a glass container lined with filter paper and filled with 50 milliliters of deionized water.

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Laatst bijgewerkt: 22 augustus 2026