This protocol is significant because it provides a specific microinjection method for an Anopheles gambiae, which has been historically difficult to transform using common genetic engineering techniques. The main advantage of this technique is that it reliably creates transgenic Anopheles gambiae. The applications of this technique extend toward novel strategies for malaria elimination by facilitating the creation of mosquitoes suitable for population suppression or modification of wild-type Anopheles gambiae populations.
This methodology can be adapted easily to different mosquito species. Our microinjection technology take patience and practice. There is a learning curve.
To begin, use an aspirator to place 20 to 30 females in a transparent 50-milliliter conical tube. Ensure the tube is cut open at both ends and is covered with latex dental film at one end and nylon mesh and filter paper at the other, where mosquitoes will lay their eggs. Place the mosquito-filled tube in a small Petri dish filled with double-distilled water, then place the tube and dish in an incubator at 28 degrees Celsius for 45 minutes.
Remove the tube from the incubator and insert the tube into an empty cage. Gently tap the tube to allow mosquitoes to fly out. Remove the tube from the cage once all the mosquitoes have flown out.
When the tube is free of mosquitoes, unscrew the bottom ring, remove the nylon, and use forceps to carefully remove the filter paper with the eggs from the tube. Place the egg-laden filter paper in a plastic Petri dish containing a layer of filter paper moistened with water. Put a membrane on a clean glass slide and use clean forceps to cover the membrane with a piece of filter paper, leaving about one millimeter of the membrane filter uncovered.
Wet the filter paper with deionized water, then use the brush to gently transfer 30 to 50 embryos to the edge of the membrane and align the eggs vertically along the membrane. Orient the eggs in the same direction so that when eggs are observed under the microinjection microscope, the posterior end is in a down position and forms an angle of 15 degrees with the needle. Line the entire edge of the membrane with eggs.
Use a microloader 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 coaxial 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. Using this protocol, an autonomous gene driven-system was inserted into the germline of a laboratory strain G3 of Anopheles gambiae.
The system was designed to target the cardinal ortholog, or Agcd, on the third chromosome in this species. The plasmid pCO37 is shown. It contains Streptococcus pyogenes Cas9 endonuclease under control of the regulatory elements of the nanos gene ortholog.
Additionally, it has a guide RNA under the control of a U6 gene promoter and the 3XP3 CFP dominant marker cassette expressing the cyan fluorescent protein. Molecular approaches verified the accurate insertion of about 10 kilo base pairs of DNA, which was designated as AgNosCd-1. Extensive followup analyses showed that AgNosCd-1 was highly efficient at drive, created few resistant alleles, and had no major genetic load due to the integration and expression of the gene drive system.
Homozygous gene drive containing mosquitoes were loss-of-function mutants, with larvae, pupae, and newly-emerged adults having a red-eye phenotype. When attempting this protocol, keeping at 88 is crucial for a good hatching to light gray eggs and avoid white eggs.
