Making a better RNAi vector for Drosophila

Use of intron spacers

Youngsik Lee, Richard W. Carthew

Research output: Contribution to journalArticle

291 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)322-329
Number of pages8
JournalMethods
Volume30
Issue number4
DOIs
Publication statusPublished - 2003 Aug 1
Externally publishedYes

Fingerprint

RNA Interference
Transgenes
Introns
Drosophila
Genes
Cloning
RNA
Gene expression
Bearings (structural)
Inverted Repeat Sequences
Organism Cloning
Double-Stranded RNA
RNA Splicing
Gene Expression
Yeast
Bacteria
Eukaryota
Drosophila melanogaster
Diptera
Tissue

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Making a better RNAi vector for Drosophila : Use of intron spacers. / Lee, Youngsik; Carthew, Richard W.

In: Methods, Vol. 30, No. 4, 01.08.2003, p. 322-329.

Research output: Contribution to journalArticle

Lee, Youngsik ; Carthew, Richard W. / Making a better RNAi vector for Drosophila : Use of intron spacers. In: Methods. 2003 ; Vol. 30, No. 4. pp. 322-329.
@article{e0ae66e4f8524a27bc0042decb347f04,
title = "Making a better RNAi vector for Drosophila: Use of intron spacers",
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.",
author = "Youngsik Lee and Carthew, {Richard W.}",
year = "2003",
month = "8",
day = "1",
doi = "10.1016/S1046-2023(03)00051-3",
language = "English",
volume = "30",
pages = "322--329",
journal = "Methods",
issn = "1046-2023",
publisher = "Academic Press Inc.",
number = "4",

}

TY - JOUR

T1 - Making a better RNAi vector for Drosophila

T2 - Use of intron spacers

AU - Lee, Youngsik

AU - Carthew, Richard W.

PY - 2003/8/1

Y1 - 2003/8/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0037711556&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037711556&partnerID=8YFLogxK

U2 - 10.1016/S1046-2023(03)00051-3

DO - 10.1016/S1046-2023(03)00051-3

M3 - Article

VL - 30

SP - 322

EP - 329

JO - Methods

JF - Methods

SN - 1046-2023

IS - 4

ER -