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Citation
Lee, Y.S., Carthew, R.W. (2003). Making a better RNAi vector for Drosophila: use of intron spacers.  Methods 30(4): 322--329.
FlyBase ID
FBrf0160727
Publication Type
Research paper
Abstract

Double-stranded RNA induces sequence-specific inhibition of gene expression at a posttranscriptional level in eukaryotes (RNAi). This natural phenomenon has been developed into a tool for studying gene function in several model organisms, including Drosophila melanogaster. Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method. We have constructed a transgene containing inverted repeats separated by a functional intron such that mRNA produced by the transgene is predicted to form loopless hairpin RNA following splicing. A single copy of the transgene effectively and uniformly silences expression of a target gene (white) in transgenic flies. We have developed a vector that is designed to produce intron-spliced hairpin RNA corresponding to any Drosophila gene. The vector is under control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. The UAS/GAL4 system allows hairpin RNA to conditionally silence gene expression in Drosophila in a tissue-specific manner. Moreover, the presence of the intron spacer greatly enhances the stability of inverted-repeat sequences in bacteria, facilitating the cloning procedure.

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    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Methods
    Title
    Methods [Supplement to Methods Enzymol.]
    Publication Year
    1990-
    ISBN/ISSN
    1046-2023
    Data From Reference
    Alleles (2)
    Genes (1)
    Molecular Constructs (2)
    Molecular Segments (15)
    Natural transposons (1)
    Insertions (4)
    Experimental Tools (2)
    Transgenic Constructs (1)