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 acti...

Full description

Saved in:
Bibliographic Details
Published in:Nucleic acids research 2001-06, Vol.29 (12), p.E55-55
Main Authors: Piccin, A, Salameh, A, Benna, C, Sandrelli, F, Mazzotta, G, Zordan, M, Rosato, E, Kyriacou, C P, Costa, R
Format: Article
Language:English
Subjects:
Animals
Animals, Genetically Modified
Crosses, Genetic
DNA-Binding Proteins
Drosophila melanogaster
Drosophila melanogaster - embryology
Drosophila melanogaster - genetics
Drosophila Proteins
Female
Fungal Proteins - genetics
Fungal Proteins - metabolism
GAL4 protein
Gene Silencing
Genetic Vectors - genetics
Insect Proteins - genetics
Male
NAR Methods Online
Nucleic Acid Conformation
Phenotype
Pigmentation - genetics
Promoter Regions, Genetic - genetics
RNA, Double-Stranded - biosynthesis
RNA, Double-Stranded - chemistry
RNA, Double-Stranded - genetics
RNA, Double-Stranded - metabolism
Saccharomyces cerevisiae Proteins
Transcription Factors - genetics
Transcription Factors - metabolism
Transformation, Genetic
Transgenes - genetics
yellow (y) gene
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1362-4962
0305-1048
1362-4962
DOI:10.1093/nar/29.12.e55