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In recent years, RNA interference (RNAi) has proven to be an effective strategy for inhibiting gene function in many organisms. This protocol describes a method for knockdown of embryonic genes in Ae. aegypti embryos that involves microinjection of small interfering RNA (siRNA) designed to target a specific gene of interest. The following procedure outlines a strategy for siRNA design, microinjection, and measure of knockdown effectiveness.
When designing siRNAs for knockdown of Ae. aegypti genes, we follow the siRNA design guidelines posted by Ambion (http://www.ambion.com/techlib/tb/tb_506.html). In general, the sequence of a gene of interest can be pasted into the Ambion siRNA target finder, which will identify 21mers that begin with an AA dinucleotide. Per Ambion’s suggestions, siRNAs should contain 30-50% GC content and lack stretches of four or more Ts or As. We recommend that multiple siRNAs be designed, as some may yield more effective knockdown than others. BLAST searches should be performed on siRNA sequences identified in order to ensure that they will only target the gene of interest. It is best to work with multiple siRNAs that yield high percentages of knockdown, as this helps to ensure that phenotypes identified are specific to the gene being targeted and not the result of off-site targeting. Negative control siRNAs typically consist of scrambled nucleotide sequences of siRNAs used for knockdown experiments. BLAST searches should be used to confirm that these control siRNAs do not target any genes in the Ae. aegypti genome. We typically order siRNAs from Dharmacon (enzymatically synthesized, A4 purity), but several other reputable vendors exist. We also order biotin-labeled siRNAs, which allows for their tracking through streptavidin staining post-injection. It should be noted that although FITC-labeled RNAs were initially utilized in our studies, autofluorescence in the embryos made them difficult to track.
Quantitative RT-PCR (qRT-PCR) is one of the most rapid methods for judging the effectiveness of siRNAs. When testing the effectiveness of new siRNAs, we typically prepare total RNA from mosquito embryos 24 - 72 hrs. post-injection. For qRT experiments, the Ae. aegypti housekeeping gene rpS17 can be used as an internal standard for data normalization (Morlais et al., 2003). In situ hybridization or immunohistochemical analysis of mRNA or protein levels can also be used to assess knockdown. These method offers a more spatial, though less quantitative, measure of knockdown.
In recent years, RNA interference (RNAi) has proven to be an effective strategy for inhibiting gene function in many organisms. Here, RNAi, which has been used successfully in mosquito larvae and adults (Blitzer et al., 2005; Luna et al., 2007), is combined with microinjection techniques used for germline transformation of Ae. aegypti (Lobo et al., 2006; Jasinskiene et al., 2007). We have used the methodology described here to knock down the Ae. aegypti frazzled (fra, Clemons et al., in preparation) and semaphorin-1a (sema1a, Haugen et al., unpublished) genes in embryos. We achieve 70% knockdown on average and have obtained up to 90% knockdown of embryonic genes. Knockdown of Ae. aegypti fra at these levels has allowed us to phenocopy the Drosophila fra mutant phenotype (Clemons et al., in preparation). Our studies suggest that this methodology can be used broadly for targeted disruption of embryonic gene function.
We are extremely grateful to Sun Longhua (Malcolm Frasier lab) who taught us to microinject mosquito embryos and has served as an expert microinjection consultant. We thank Frank Collins for advice, encouragement, and use of equipment. Thanks to Alexandra Tobler for comments on the protocol. Development of the methodology described here was funded by NIH/NIAID Award R01 AI 081795-01 and an IUSM Research Support Funds Grant (both to MDS).
|MICROINJECTION BUFFER (10x)|
|Reagent||Quantity (for 500 ml)||Final concentration|
|KCl||1.86 g||50 mM|
|NaH2PO4 (anhydrous)||0.06 g||1 mM|
Bring volume to 500 ml with sterile RNAse-free dH20. Adjust the pH to 7.2 and store at room temperature.
Conflicts of interest: none declared