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Nucleic Acids Research, 2001, Vol. 29, No. 12 e55
© 2001 Oxford University Press

Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer

Alberto Piccin*, Ahmad Salameh, Clara Benna, Federica Sandrelli, Gabriella Mazzotta, Mauro Zordan, Ezio Rosato1, Charalambos P. Kyriacou2 and Rodolfo Costa

Dipartimento di Biologia, Università di Padova, via Ugo Bassi 58/B, 35131 Padova, Italy, 1Department of Biology and 2Department of Genetics, University of Leicester, Leicester LE1 7RH, UK

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.

* To whom correspondence should be addressed. Tel: +39 049 827 6228; Fax: +39 049 827 6209; Email: costast4{at}civ.bio.unipd.it


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