Injection of dsRNA into Female A. aegypti Mosquitos

Hi, my name's Brian Luna. I'm here today at the University of California Irvine from the James Lab. Today I'll be talking to you about double strated, RNA injections into adult eighties gyp type female mosquitoes.

So the procedure consists of, of using a manual setup and injecting the adult females into the, into the thorax.Double-stranded. RNA injections can be used to knock down numerous genes in our lab. Specifically, we're using it to knock down genes related to steroid biosynthesis.

Now I'm gonna be talking to you about the preparation of the needles. Now I prepare the needles using these foal silicate glass capillary tubes. Now these, these capillaries are pulled on our Sutter micro injection puller, and the next step after that the needles have been pulled is to calibrate them using water.

The next step in this process is the calibration of the needles. So once the needles have been pulled from the Sutter Polar, they come out looking like, so the final product will have the needles clearly marked with a black sharpie marker. Now, in order to to standardize the markings, each needle is loaded with a set amount of volume by a micro pipetter.

Calibration of the needles is, is important, or else we will not know the exact concentration or volume that we are injecting into each mosquito. For the purpose of this demonstration, I've loaded the capillary tubes with a food coloring dye. Instead of a typical double stranded, RNA suspension, the doublet stranded, RNA, can be loaded into the needles by inserting the needles into the solution and capillary action will drop, drop the fluid.

Now I'm gonna be talking to you about our injection setup. It's composed of a 50 milliliter syringe, a stock cock assembly attached to a rubber hose and a glass capillary adapter. The stock cock assembly can be, can be adjusted to three separate positions.

This first position is the resting position. The second position is in injection position, and the third position is used for filling the syringe with air, for the pressure to build the pressure for the injection. So during injection, the mosquitoes are kept on ice to keep them asleep.

After injection, the best thing to do is to keep put em back at room temperature to increase the survivability. So this is done just using an or ordinary cup such as this and a thousand microliter pipette. The tip of the pipette is cut off and then inserted into the cup, and the final product looks like this.

On top of the cup, we place a, a mesh netting and a lid cover. And now this just allows for air to travel in and outta the cups and keep some mosquitoes from flying away. The mosquitoes will not fly out of the pipette tip that was inserted into the cup.

Now we're almost ready for the injections. With the double strand RNA. The next step is to anesthetize the mosquitoes using CO2 and to place the mosquitoes on ice to keep them asleep.

Now we're ready to actually, the mosquitoes. The first step of this process is to fill, fill the syringe with air. Once this is done, the valve is adjusted to the resting position.

So here I am about to insert the needle into the thorax of the mosquito. The next step of this process is to rotate the valve to the injection position and inject the desired amount of volume. And once the injection is complete, return the mosquito to the cup.

I've just finished showing you the technique for injecting double strand of RNA into adult eighties gti. Mosquitoes knockdown can be determined by either using R-T-P-C-R or quantitative R-T-P-C-R. I'll be using R-T-P-C-R to determine knockdown efficiency over a specific time point of my g of Interest.