Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer.
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.
Piccin
A
A
Dipartimento di Biologia, Università di Padova, via Ugo Bassi 58/B, 35131 Padova, Italy.
Salameh
A
A
Benna
C
C
Sandrelli
F
F
Mazzotta
G
G
Zordan
M
M
Rosato
E
E
Kyriacou
C P
CP
Costa
R
R
D000818:Animals; D030801:Animals, Genetically Modified; D003433:Crosses, Genetic; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D005260:Female; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D019476:Insect Proteins / Q000235:genetics*; D008297:Male; D009690:Nucleic Acid Conformation; D010641:Phenotype; D010858:Pigmentation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012330:RNA, Double-Stranded / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D019476:Insect Proteins; D012330:RNA, Double-Stranded; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C050680:y protein, Drosophila