Kafatos:Anopheles gambiae dsRNA Gene KD and Midgut Dissection
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Anopheles gambiae dsRNA Gene KD and Midgut Dissection
1. Inject mosquitoes (not >48h post-eclosion) with dsRNA from in vitro transcription reaction.
- It is desirable to inject enough individuals to account for death due to dthe injection process, age and those who will not blood-feed sufficiently well to establish an infection.
- Note: care should be taken to schedule the feeding of your mosquitoes at this time => infection of mice and day of feeding needs to be taken into account.
2. Wait 4 days for the knockout to take effect.
3. Feed surviving injected mosquitoes on infected mice.
- 30 min feeding is generally sufficient, 45 min is better.
4. After feeding, incubate mosquitoes in a humid box or bag at 20˚ C.
5. Select out non-blood-fed individuals <48h post infection on ice.
6. Wait until 5 to 10 days (ideally 7d) post-infection to perform dissections, in order to allow oocysts to begin to develop.
7. Knockdown surviving individuals from a cohort in a Falcon Tube on ice.
8. Perform dissections in a dissection dish in ~1 ml PBS or on a glass slide in PBS on ice.
- Fine, sharp foreceps are critical for the dissections. Hold the animal in place with one pair of foreceps at the thorax, and one pair holding the last abdominal segment. Holding the anterior end in place, gently pull away with the posterior until the gut is released from the abdominal cavity.
9. Immediately place the gut in a dissection dish with 4% formaldehyde for at least 30 min to 1 hour to fix, rocking gently.
- Formaldehyde should be prepared fresh for each use, diluted in PBS from the stock concentration of 16% (can be kept at -20˚)
10. Wash fixed guts 3x 15min in PBS, rocking gently.
- Guts can be washed for over an hour, if desired, without harm (wrap in Aluminum foil to shield from light, if you are using the GFP parasite).
11. Mount washed guts in 1 drop of Vecta Shield fluorescence preservativeto on a glass slide and seal coverslip with nail polish.
- Ideally, results should be examined immediately, fluorescence is weaker the following day. If have to, keep the slides ON in the dark at 4˚ C.