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RNAi Interference by dsRNA Injection into Drosophila Embryos
Published on: April 11, 2011
Larval RNAi in Drosophila?
Sherry C Miller1, Susan J Brown, Yoshinori Tomoyasu
1Division of Biology, Kansas State University, Manhattan, KS 66506, USA.
Development Genes and Evolution
|July 30, 2008
Summary
Most Drosophila tissues cannot uptake extracellular double-stranded RNA (dsRNA) to trigger RNA interference (RNAi), unlike hemocytes and most Tribolium tissues. This study investigates insect dsRNA uptake mechanisms for gene knockdown applications.
Area of Science:
- Molecular Biology
- Genetics
- Entomology
Background:
- RNA interference (RNAi) is a key gene knockdown technique requiring cellular uptake of double-stranded RNA (dsRNA).
- Systemic RNAi, where cells uptake extracellular dsRNA, is known in organisms like C. elegans.
- The ability of Drosophila melanogaster cells to uptake extracellular dsRNA is widely assumed to be limited, but lacks extensive data.
Purpose of the Study:
- To investigate the capacity of Drosophila melanogaster tissues to uptake extracellular dsRNA and initiate RNAi.
- To compare dsRNA uptake and RNAi response in Drosophila melanogaster with that of Tribolium castaneum, an insect with known systemic RNAi.
- To clarify the tissue-specific accessibility of RNAi in insects.
Main Methods:
- Utilized Gal4/upstream activation sequence (UAS) transgenic flies and the mosaic analysis with a repressible cell marker (MARCM) system in Drosophila melanogaster.
- Performed extracellular injection of dsRNA into Drosophila melanogaster larvae.
- Overexpressed dsRNA intracellularly using hairpin RNAs in Drosophila melanogaster tissues.
- Examined dsRNA response in various tissues of Tribolium castaneum.
Main Results:
- Extracellular dsRNA injection failed to trigger RNAi in most Drosophila melanogaster tissues, except for hemocytes.
- Drosophila melanogaster tissues possess functional RNAi machinery, as demonstrated by efficient knockdown using intracellular hairpin RNAs.
- Virtually all Tribolium castaneum tissues exhibited a robust response to extracellular dsRNA, contrasting with Drosophila melanogaster.
- Hemocytes in Drosophila melanogaster were identified as an exception capable of responding to extracellular dsRNA.
Conclusions:
- Most Drosophila melanogaster tissues lack the ability to uptake extracellular dsRNA for RNAi, contrary to common belief.
- Drosophila melanogaster hemocytes represent a specific tissue type capable of initiating RNAi from extracellular dsRNA.
- Tribolium castaneum demonstrates a broad capacity for systemic RNAi across most tissues, differing significantly from Drosophila melanogaster.
- Findings provide crucial insights into insect-specific mechanisms of dsRNA uptake and systemic RNAi, aiding future gene knockdown strategies.
