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Title: Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer
Authors: Piccin, A.
Salameh, A.
Benna, C.
Sandrelli, F.
Mazzotta, G.
Zordan, Mauro A.
Rosato, Ezio
Kyriacou, Charalambos P.
Costa, R.
First Published: Dec-2001
Citation: Nucleic Acids Research, 2001, 29 (12) : e55
Abstract: We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y‐IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS–y–IR hairpin is expressed under the control of different promoter–GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.
DOI Link: 10.1093/nar/29.12.e55
ISSN: 0305-1048
eISSN: 1362-4962
Version: Publisher Version
Status: Peer-reviewed
Type: Article
Rights: Copyright © 2001, Oxford University Press (OUP). Deposited with reference to the publisher’s open access archiving policy.
Appears in Collections:Published Articles, Dept. of Genetics

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