RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.
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Method Article
RNAi Interference by dsRNA Injection into Drosophila Embryos
In This Article
Summary
Abstract
Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.
Protocol
1. Embryo Collection
- Set up cages at 25°C using 2-4 day-old w1118 flies.Grape juice plates are changed every hour during the day to synchronize the egg collection over 1-2 day period before collection
- Collect embryos for 1hr at 25°C
- Cut a rectangular piece of grape juice agar, cut lightly in the middle with a razor blade, leave a line in the agar
- Use a metal probe to transfer embryos from grape juice plate to your piece of grape juice agar, line them up in a straight line with the embryo's longer axis at 45 degree angle to the line in the middle of the agar. Get a straight line of about 50 embryos. Make sure all the em....
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Discussion
The dsRNA injection method present here enables a very sensitive and rapid analysis of gene function in Drosophila tracheal development. This method can potentially be applied to analyze gene function for other tissue and organ development.
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Disclosures
No conflicts of interest declared.
Acknowledgements
The authors would like to thank Stephen Crews for dysfusion cDNA, Dys antibody and w1118 flies.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Halocarbon oil 700 | Sigma-Aldrich | H8898 | |
| Picospritzer III picopump | Parker Hannifin Corporation | 051-0500-900 | |
| Micro-pipettes | Fisher Scientific | 21170M | |
| Microloaders | Eppendorf | 930001007 |
References
- St Johnston, D., Nusslein-Volhard, C. The origin of pattern and polarity in the Drosophila embryo. Cell. 68, 201-219 (1992).
- Venken, K. J., Bellen, H. J. Emerging technologies for gene manipulation in Drosophila melanoga....
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