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


RESULTS
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
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.
Langer
Christoph C H
CC
Max-Planck-Institute of Biochemistry, Martinsried, Germany.
Ejsmont
Radoslaw K
RK
Schönbauer
Cornelia
C
Schnorrer
Frank
F
Tomancak
Pavel
P
D000818:Animals; D001483:Base Sequence; D004330:Drosophila / Q000235:genetics*; D055785:Gene Knockdown Techniques; D023281:Genomics; D008969:Molecular Sequence Data; D034622:RNA Interference; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D019076:Transgenes