Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.
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Method Article
Injection of dsRNA into Female A. aegypti Mosquitos
In This Article
Summary
Abstract
Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.
Protocol
- Make microinjection needles by pulling glass capillary tubes. We use a needle puller from Sutter Instrument CO, (Model P-2000). The needle puller program specifications are:
heat: 275
fil: 3
vel: 37
del: 250
pul: 125 - Load a known volume (e.g., 1 μl) of water in the needles and manually mark the scale with a fine sharpie.
- Set up a post-injection cup for the mosquitoes. First cut the outmost tip off of a 1000 μl pipette tip. Then, cut a hole in the side of a cup and insert the remaining part of the pipette tip in the cup. Put a net or lid on top of the cup.
- Fill two Styrofoam boxes with ice. Place ice box....
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Disclosures
The authors have nothing to disclose.
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