Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Every 15 seconds, there is a child dying from malaria in the world, in here in the James lab, or trying To make French mosquitoes that cannot carry the malaria persons. My name is TIUs. I'm here at Universal of California Irvine at Department of Molecular Biology and Biochemistry in the lab of Anthony James.

Today I will show you how to make transgenic and not Stevens eye. And we are doing this to make malaria mosquitoes refractory so they cannot Carry the malaria parasite. Now I have prepared a vial with Isotonic buffer where the mosquitoes are going to lay their eggs.

I'm gonna use this aspirator to suck up the mosquitoes and put in a vial. This vial is then going into an incubator that is 30 degrees Celsius, and we'll leave it for about one hour in a cup. Now we're gonna pull the microinjection needles.

These are needles made of quartz and we have to use a very fine tip. Therefore, we need these needle puller. We use a needle puller from Suter instruments and also the needles from subtle in SU instruments.

So now taking out the mosquitoes and I will put these eggs down into the Petri dish where I have a filter paper soaked in isotonic buffer that is just to keep the embryos wet during the injection period. The amount of water on the filter paper is critical. You don't want the eggs to float around and you don't want it to get dry.

Now I'm gonna move the embryo from the egg paper onto another three MM watman paper that has been soaked in isotonic buffer. Now do this to take away the Corian before aligning them up for the microinjection. So now I'm gonna take away the Corian, which is this silvery surface on the eggs.

And I do that by brushing them off with my brush. So on top here we have an embryo where the Corian is still sitting and they have this silvery shining part. Where is the one below?

I have been successful to taking out the Corian and this the top one will probably slip on a tape, whereas the bottom one will stick. And that is what's important when you do an off even side. Now I am aligning the embryos.

The embryos are factor or bigger on the anterior end, and that one I'm placing to the right. On the left I have the poster end, which is slimmer and which I, where I will inject the embryos. I'm trying to put them together so they help each other to stay in position.

And usually I do about 10 in a line together. Now I have a line, the embryos, and I will transfer them to a cover slip. On the cover slip.

I put wig tape in order for them to stick. When I'm done that I will dry the filter paper where the embryos are sitting with another filter paper, put the embryos on the cover slip and let it dry for 30 seconds before I apply oil. Now we have aligned the embryos and have them under the oil.

On the microscope stage, we will inject DNA mixed with food dye using this special tip of the microbe pattern And inject this one into the port's needles. Now we have filled the needle and put it back on a micro injector. And now we're ready To do the micro injections.

Now I'm moving in the needle to sharpen it and I, so I can see it focused. Once that is done, I move a weight needle and I move in the embryos and focus those as well. And when that is done, I'm moving the needle again until I have it in position and I move on my embryos.

Now when the needle gets through the oil, it gets blurry and you need to adjust it again. So that's exactly shark. Exactly, shark tick.

And now I'm ready to do the injections. You could see that I touched the embryo with the needle, and by doing that I broke the tip and you can see a small droplet coming out that would be injected into the embryo. So here inject and get it out again.

And I check every time between each embryo that I have a small droplet coming out to the tip of the needle. And as you can see, I need to readjust for each embryo to get in perfect position. Now this one was leaking.

As you can see, when I do my second injection out in the oil, the bubble becomes very large and I need to readjust the pressure of the micro injector. So now we're done with the micro injections and we're going to move the embryos from the microscope into the Petri dish, which we have filled with isotonic buffer. I will wipe off the oil from the cover slip and cut it into a small piece, which I put down into the Petri dish.

Now we're gonna leave them here until the end of the day and room temperature. And then we move Them down to into the insectory. Today I've shown you how to inject an awfully Steven side To make transient mosquitoes.

And now we just have to wait for the larvae to hatch all the larva to hatch. We will bring up to adults and then they will mate. And after that we're gonna have three weeks before we can start a screen if they're Refactor or not.