Cross-Species RNAi Rescue Platform in Drosophila melanogaster
41965 GEN18331165.pdf (2.659Mb)
Access StatusFull text of the requested work is not available in DASH at this time ("restricted access"). For more information on restricted deposits, see our FAQ.
MetadataShow full item record
CitationKondo, Shu, Matthew Booker, and Norbert Perrimon. 2009. “Cross-Species RNAi Rescue Platform inDrosophila Melanogaster.” Genetics 183 (3): 1165–73. https://doi.org/10.1534/genetics.109.106567.
AbstractRNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41384479
- HMS Scholarly Articles