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. 2001 Jun 15;29(12):E55-5.
doi: 10.1093/nar/29.12.e55.

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

A Piccin  1 , A Salameh, C Benna, F Sandrelli, G Mazzotta, M Zordan, E Rosato, C P Kyriacou, R Costa
Affiliations

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

A Piccin et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
Scheme for the generation of transgenic, hairpin-mediated RNAi. A coding fragment of the yellow gene was cloned as an inverted repeat into the Drosophila transformation vector pUAST, using a GFP fragment to separate the repeats and therefore facilitate the cloning. The resulting transformants were then crossed to various GAL4 strains to drive expression of the hairpin-encoding transgene.
Figure 2
Figure 2
Effect of UAS–y–IR-encoded dsRNA transcription on the pigmentation of male and female adult D.melanogaster. y+ flies (w1118; da-GAL4/+) and y1-type mutants are shown for comparison. Expression of UAS–y–IR-encoded dsRNA mediated by a daughterless–GAL4 driver blocks melanin pigment deposition in cuticle and wing structures but not in bristles, mimicking the colour pattern of y2-type mutants. Act5c–GAL4 driven dsRNA transcription triggers effective RNAi also in bristles, producing y1-like phenocopies.
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