In vivo RNAi rescue in Drosophila melanogaster with genomic transgenes from Drosophila pseudoobscura.
| Autor: | Langer CC; Max-Planck-Institute of Biochemistry, Martinsried, Germany., Ejsmont RK, Schönbauer C, Schnorrer F, Tomancak P |
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| Jazyk: | angličtina |
| Zdroj: | PloS one [PLoS One] 2010 Jan 28; Vol. 5 (1), pp. e8928. Date of Electronic Publication: 2010 Jan 28. |
| DOI: | 10.1371/journal.pone.0008928 |
| Abstrakt: | 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. |
| Databáze: | MEDLINE |
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