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PLoS One. 2010 Jan 28;5(1):e8928. doi: 10.1371/journal.pone.0008928.

In vivo RNAi rescue in Drosophila melanogaster with genomic transgenes from Drosophila pseudoobscura.

Author information

1
Max-Planck-Institute of Biochemistry, Martinsried, Germany.

Abstract

BACKGROUND:

Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

METHODOLOGY/PRINCIPAL FINDINGS:

We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

CONCLUSIONS/SIGNIFICANCE:

The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

PMID:
20126626
PMCID:
PMC2812509
DOI:
10.1371/journal.pone.0008928
[Indexed for MEDLINE]
Free PMC Article
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