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 Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.
RNAi-mediated gene silencing in mosquitoes requires prior preparation and purification of specific dsRNAs that target the gene of interest. For dsRNA synthesis, we recommend the Ambion Megascript kit which makes use of a T7 RNA polymerase-mediated in vitro transcription reaction. For dsRNA purification, we recommend the Qiagen RNeasy kit. Samples of dsRNA should be quantified and adjusted to 3 μg/μL in water. Other materials you will need include: fine-tipped forceps, glass slide, glass microcapillary needles, microinjector (we use Drummond’s Nanoject II), a light microscope mounted above a cold block, a covered glass Petri dish, paper towels and a bucket of ice. You will also need a way to collect mosquitoes and suitable container for them along with a food source such as cotton ball soaked in 10% sucrose.
Note: Efficiency of gene silencing is highly variable and depends on a number of factors including transcript and protein turn-over rates, and dsRNA uptake efficiency by cells and organs. We have found that silencing begins as early as 1 day post injection, and can last up to 6 days post injection. Use transcript and protein detection methods such as quantitative PCR and Western blotting to validate silencing.
Note: There are several variations on several steps of the mosquito injection and RNAi-mediated gene silencing technique. Some variations are described by: Boisson, et al. (2006) and Blandin, et al.(2002).
Efficiency of gene silencing is highly variable and depends on a number of factors including transcript and protein turn-over rates, and dsRNA uptake efficiency by cells and organs. We have found that silencing begins as early as 1 day post injection and can last up to 6 days post injection. Use transcript and protein detection methods such as quantitative PCR and Western blotting to validate silencing.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Megascript | kit | Ambion | for dsRNA synthesis; T7 RNA polymerase-mediated in vitro transcription reaction | |
RNeasy | Qiagen | For dsRNA purification | ||
dsRNA | should be quantified and adjusted to 3 μg/ μL in water | |||
fine-tipped forceps | ||||
glass slide | ||||
glass microcapillary needles | ||||
microinjector | Drummond | Nanoject II | ||
light microscope | mounted above a cold block | |||
Petri dish | covered, glass | |||
paper towels | ||||
10% sucrose | food | |||
A. gambiae | Animal | mosquitos |