Validating RNAi phenotypes in Drosophila using a synthetic RNAi-resistant transgene

RNA interference (RNAi) is a powerful and widely used approach to investigate gene function, but a major limitation of the approach is the high incidence of non-specific phenotypes that arise due to off-target effects. We previously showed that RNAi-mediated knock-down of pico, which encodes the onl...

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Bibliographic Details
Published in:PloS one 2013-08, Vol.8 (8), p.e70489-e70489
Main Authors: Jonchere, Vincent, Bennett, Daimark
Format: Article
Language:English
Subjects:
Adapters
Adaptor proteins
Animal genetics
Animals
Base Sequence
Biology
Cell cycle
Cell growth
Cell number
Cell size
Double-stranded RNA
Drosophila
Drosophila - genetics
Drosophila melanogaster
Evacuations & rescues
Experiments
Gene expression
Genes
Genetic aspects
Genetic engineering
Genetics
Genomes
Homology
Insects
Life Sciences
Male
Molecular Sequence Data
Morphology
Phenotype
Proteins
Ribonucleic acid
RNA
RNA Interference
RNA, Small Interfering - genetics
RNA-mediated interference
Signal transduction
Transgenes
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Summary:RNA interference (RNAi) is a powerful and widely used approach to investigate gene function, but a major limitation of the approach is the high incidence of non-specific phenotypes that arise due to off-target effects. We previously showed that RNAi-mediated knock-down of pico, which encodes the only member of the MRL family of adapter proteins in Drosophila, resulted in reduction in cell number and size leading to reduced tissue growth. In contrast, a recent study reported that pico knockdown leads to tissue dysmorphology, pointing to an indirect role for pico in the control of wing size. To understand the cause of this disparity we have utilised a synthetic RNAi-resistant transgene, which bears minimal sequence homology to the predicted dsRNA but encodes wild type Pico protein, to reanalyse the RNAi lines used in the two studies. We find that the RNAi lines from different sources exhibit different effects, with one set of lines uniquely resulting in a tissue dysmorphology phenotype when expressed in the developing wing. Importantly, the loss of tissue morphology fails to be complemented by co-overexpression of RNAi-resistant pico suggesting that this phenotype is the result of an off-target effect. This highlights the importance of careful validation of RNAi-induced phenotypes, and shows the potential of synthetic transgenes for their experimental validation.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0070489