Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

I am Lindsay Garver and I'm a PhD student in the lab of Dr.George Dimopoulos at the Johns Hopkins School of Public Health. And I'm gonna be showing you how to do RNAi mediated silencing of Genes using an injection technique. So the first step is to collect The mosquitoes that we're going to be injecting.

These are just stock an awfully Gabi. It's a mixture of males and females. And this is our battery powered aspirator.

It has a fan inside, which will create suction, drawing the mosquitoes up through this nozzle into this reservoir. So we unplug. We have a small access door, which is typically taped to keep the mosquitoes inside, but we open it and insert the nozzle, turn it on and use to collect.

Once we have the mosquitoes inside, we plug the top of the nozzle, see the mosquitoes there in the reservoir. Then we want to anesthetize these mosquitoes. So we bury this reservoir in an ice bucket we cover with ice and wait for The mosquitoes to go down.

Okay, the next step is to inject the Mosquitoes and to use that, we'll be using this, which is a nano jet two. It consists of a wand portion, which I'll be holding. And this will contain the needle, which will dispense the liquid into the mosquito.

The control panel, which has our fill and empty buttons and is responsible for drawing liquid into and out of the needle. And then this is the footed. This will be put on the floor.

And pushing this with my foot will dispense one drop of the appropriate volume into the mosquito. So while the mosquitoes are on ice, we prepare the workstation by setting a cold block to zero degrees to two degrees Celsius. This cold block has a flat attachment, which then has a glass slide mounted on it.

And this will keep the mosquitoes from moving while we're trying to manipulate them. I will also take one of these glass capillary needles. This is just a regular glass capillary tube that has been heated and pulled in the middle to create a needle.

And this is what will hold our liquid. And then this is what we'll use to inject the mosquito and dispense the liquid. So the next thing we have to do is to place the capillary needle onto our injecting wand.

We remove the nose piece. Make sure that the interior rings are situated according to your manufacturer's instructions. Slide the glass needle down into the nose piece, being careful of the tip so that you don't break it.

Once that's secure, slide the needle of the wand into the capillary needle and screw the nose piece on tightly. The next thing is to make sure that the needle of your capillary tube is a good width for injections. This is a really important point because too thin of a needle will not dispense the correct amount of liquid and too thick of a needle point will injure the mosquito and possibly cause death.

This is something that takes a lot of practice, but a, a good width is actually crucial for proper injection and survival of your mosquitoes. What I do is to use your fine tipped forceps to break the tip of the needle such that the needle is still compliant yet stable. We need our double stranded RNA sample.

This sample was prepared using a kit. There are a variety of kits on the market that can generate specific double standard RNAs to the gene of interest. We use our RNAs at a concentration of three micrograms per microliter, and we have our nano inject set so that one injection dispenses 69 nanoliters position.

The tube put our needle into the liquid and use the fill button on the n eject to draw liquid into the needle. The amount of liquid that you'll need in the needle is directly proportional to the number of mosquitoes you'll be injecting.Okay? And we now need to prepare mosquitoes.

So we take our plastic P two dish and we embed it in our bucket of ice. We check that our mosquitoes are not moving and we spread them out thinly on the cold Petri dish. This ensures that as we're working, the mosquitoes are not moving and they're not flying around.

We then position the mosquitoes on our glass slide, which is now at zero degrees Celsius. And the positioning of the mosquitoes is crucial for how we inject them. So you want your mosquitoes to be lined up orderly so that you know which have been injected and which have not been injected.

And you also want there to be sufficient space between your mosquitoes so that you can reach them with the injector. So we transfer individual mosquitoes from the peewee dish to the slide, taking care, taking care to handle them by their legs. This avoids squishing the bodies and it results in greater survival.

So now we're ready to inject. We use the fine tip forceps to support the mosquito on one side, I'm right-handed, so I use this in my left hand. And then we inject from the other side inserting the tip of the needle into the side of the thorax, such that the thorax of the mosquito is sandwiched between the tips of the forceps and the needle point and the needle point enters.

Once the needle point is inside the thorax, push the foot pedal, you'll hear a beep and liquid will be dispensed into the mosquito thorax. Then you move to the next one and repeat. Once in a while when you withdraw the needle, you'll see a drop of liquid that has exited the entry site.

And if this happens, then you can wait for the liquid to be reabsorbed into the entry site. If that happens, that's fine. If the drop is not reabsorbed, if the drop remains on the outside of the thorax, then that mosquito must be discarded and you, you inject another one in its place.

It is also important to make sure that your needle only goes as far into the thorax as needed. You don't want to poke through to the other side or to allow too much width of needle to go through. The thorax or mosquito survival will be compromised.

So we continue that procedure For all of the mosquitoes as needed. So to store your injected Mosquitoes, we used a one pint cardboard wax lined cup that has been covered with a thin mesh netting and secured with a rubber band and a cardboard lid. We opened the access door that is covered with tape and carefully add our mosquitoes To help your mosquitoes survive.

You should add a cotton ball that has been wedded with a 10%sucrose solution that acts as a food source for the mosquitoes while they're in here. And a moist folded paper towel. Since wounded mosquitoes do have a tendency to dry out, this will help preserve the humidity and aid survival of your injected mosquitoes.

We top this off with a Petri lid to contain the the moisture so that our paper towel and our cotton balls stay wet. And then we incubate this in under normal environmentally controlled conditions. So the molecular kinetics of RNAi mediated gene silencing vary quite a bit from gene to gene.

And so for some genes, the silencing can take place within a day and for other genes, efficient silencing won't take place for several days. Differences between each gene can be measured using quantitative real-time PCR, and this will monitor the transcript levels within your mosquitoes and will tell you when you have achieved efficient silencing. So I would recommend setting aside a couple of your injected mosquitoes for that purpose.

This also will help you determine how efficient your double stranded RNAs are in general, whether you, your specific double stranded RNAs can achieve efficient silencing. And if you are in fact Interfering with transcript levels, I've just shown you the procedure for RNAi mediated Gene silencing of anomaly Gambia mosquitoes. This procedure involves the micro injection of doublet stranded RNAs into the thorax of Anole Gabi mosquitoes.

This procedure is important because it is one of the easiest and most effective waste to manipulate individual genes In mosquitoes.