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The overall goal of this method is the delivery of RNA interference triggers in Anopheles gambiae to initiate gene knockdowns that begin during the pupal stage of development. This method can help answer key questions in the field of vector biology and uncover genes that play a role during pre-adult or early adult developmental stages. The main advantage of this technique is that it provides researchers with an opportunity to perform rapid functional genomic studies, beginning gene knockdown during the developmental interval, the pupal stage, when RNA interference injection protocols have not been implemented in the past.

Visual demonstration of this method is critical because the injection steps can be difficult to master due to the precision required and the delicate nature of pupae during this developmental stage. After selecting the gene of interest, identify a 200 to 800 base pair region that is predicted to have no off target effects and perform standard PCR amplification to obtain double-stranded RNA synthesis template flanked by a T7 promoter sequence. Then synthesize the double-stranded RNA and clean up the products using a commercial in vitro kit.

Adjust the final RNA concentration to three micrograms per microliter. Next, use a standard 2%agarose gel to compare one half microliter of the double-stranded RNA with template DNA and standard markers. Once run out, check the double-stranded RNA band quality and length.

There should be no visible non-specific products. The double-stranded RNA will migrate more slowly than template DNA. For storage, prepare double-stranded RNA aliquots, and keep them at minus 20 degrees Celsius.

Begin with preparing the Fast Green FCF dye. First, dilute the dye to 0.1%volume by volume in RNase-Free water. Then, transfer one microliter volumes of the preparation to 1.5 milliliter microfuge tubes.

Heat the loaded tubes at 65 degrees Celsius for about three hours to evaporate the water. Then cool the tubes for at least 30 minutes before using the dye solids. Next, prepare pulled glass injection needles from borosilicate glass tubes.

Set the needle puller for a tip diameter of 10 to 30 microns. For a NARISHIGE capillary puller, set the first heater to 100, and the second heater to 70. Then clamp in the capillary needle, and proceed with pulling.

Now, prepare the injection station. Select a platform that can be easily maneuvered under the microscope. On the platform, first tape down a thick filter paper.

Then, onto the thick filter paper, tape down one thin filter paper. Next, to the dye solid tubes, add 10 microliters of each double-stranded RNA to be injected, and re-suspend the dye. Store these tubes on ice.

Now, collect the pupae to be injected. Fill a 60 millimeter petri dish with 10 milliliters of deionized water, and transfer about 50 pale-colored pupae into the dish, using a plastic transfer pipette. Use only lightly pigmented pupae, as pupae that have already reached medium to dark tanning levels will suffer cuticle damage, and have poor survival rates.

The first step to the microinjection is to open the needle. Under the microscope, break off the very distal tip of the needle using fine forceps. Then backfill the needle with mineral oil using a 30 gauge needle and syringe.

Once filled, expel the excess oil using the microinjector. Set the microinjector to pull 69 nanoliter volumes. Then, front fill the needle with the prepared injection RNA.

Test the dispensing of double-stranded RNA for problems. Blockage in the tip and inadequately secured needles are the main sources of problems. Once the injector is prepared, pick one to three pupae and transfer them to the filter paper stage.

Using a paintbrush, orient the pupa so that the dorsal cuticle is accessible. By pressing on the paper, the excess water on the pupae will be absorbed. While keeping the pupa still with a wet paintbrush, use an anterior to posterior motion to insert the needle into its dorsal cuticle between the abdomen and the thorax, at approximately a 30 degree angle.

Then, inject one to two pulses of double-stranded RNA into the hemolymph. Dispersion of the dye throughout the body should be visible if the injection has succeeded. While injecting the pupa, it's critical that the needle only punctures the dorsal cuticle.

If the needle puncture extends through the ventral cuticle, pooling of dye-labeled liquid will be evident on the exterior of the pupa, or the dye will transfer onto the filter paper. In such cases, the pupa must be discarded. If dye does not distribute throughout the hemolymph, the tip is most likely obstructed.

Move the tip slightly and determine whether any release of RNA and dye has occurred. If there is no indication of dye release, discard the pupa. Then, assess the tip of the needle and perform another injection on a new pupa.

After successfully injecting a pupa, use the wet paintbrush to gently release it from the needle, and move it into the dish of water where it will complete development. Place the dish of injected pupae in a mosquito cage or container under normal rearing conditions. For an adult food source, provide glucose in a 10%weight by volume solution soaked into a cotton ball.

After the adults have emerged, compare them with controls. For example, double-stranded SRPN2 insects should be assessed daily for pseudotumor formation. To do this, chill the adults for two to three minutes at minus 20 degrees Celsius, and transfer them to a two degree Celsius cold plate for viewing.

After viewing the adults, return them to the insectary rearing area. Once the technique was mastered, injected pupae emerged with a frequency of about 70%In a subset of the injected pupae, partial emergence from the pupal case occurred, leading to inviable mosquitoes. There was no delay in emergence rates between injected and un-injected mosquitoes, and the impacts of injection did not vary by gender.

Ten days after emergence, there was no notable change in survivorship of the injected mosquitoes compared to the non-injected controls. The serine protease inhibitor, SRPN2, was used as a positive control, and double-stranded LacZ was used as a negative control for double-stranded RNA injections. By three days post-emergence, pseudotumors in the double-stranded SRPN2 injected adults were evident via light microscopy.

After eight days, pseudotumors were very prominent. In adult stage mosquitoes, melanotic pseudotumors were observable through the cuticle of most double-stranded SRPN2 injected animals, while no pseudotumors were observable in controls. Five days post injection, SRPN2 protein levels were significantly lower in double-stranded SRPN2 injected mosquitoes, compared to double-stranded LacZ injected controls and non-injected controls.

After watching this video, you should have a good understanding of how to perform RNA interference trigger delivery into Anopheles gambiae pupae, using this newly developed injection protocol. Generally, individuals new to this method will struggle because of the unpredictable movement of the pupa. However, practicing the correct positioning and stabilization of the pupa greatly enhances the rate of successful injection, as well as injection quality.

Once you've mastered the technique, these injections require approximately 30 seconds per pupa. With the development of this technique, we have paved the way for researchers in the field of vector biology to explore and assess gene function during pre-adult and early adult developmental stages in the malaria vector Anopheles gambiae. Following this procedure, other methods such as Western blot analysis or quantitative PCR can be performed to determine the extent of target gene knockdown at the level of protein or transcript expression, respectively.

Ultimately, this technique can help identify genes that are critical for parasite transmission by mosquitoes, and this approach will help us identify new targets and provide new insights into how we can create more effective vector control strategies.