May 3, 2003

Comparative analysis of DNA vectors at mediating RNAi in Anopheles mosquito cells and larvae

The Journal of Experimental Biology
Anthony E BrownFlaminia Catteruccia

Abstract

Heritable RNA interference (RNAi) mediated by transgenes exhibiting dyad symmetry represents an important tool to study the function of genes expressed at late developmental stages. In this study, we determined whether the transcriptional machinery of Anopheles mosquitoes is capable of directing suppression of gene expression from DNA constructs designed to transcribe double-stranded RNA (dsRNA) as extended hairpin-loop RNAs. A series of DNA vectors containing sense and antisense regions of the green fluorescent protein EGFP target gene was developed. The effect of these vectors on a transiently expressed or stably integrated EGFP gene was assessed in an Anopheles gambiae cell line and in Anopheles stephensi larvae. Our data indicate that dsRNA-mediated silencing of a target gene from plasmid DNA can be achieved at high levels in Anopheles cell lines and larvae. The region that links the sense and antisense sequences of the target gene plays a determining role in the degree of silencing observed. These results provide important information for the development of heritable RNAi in Anopheles.

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Mentioned in this Paper

Shuttle Vectors
Cosuppression
RNA, Double-Stranded
Transcription, Genetic
Candidate Disease Gene
Immunofluorescence Microscopy
Gene Expression
Larva
Visual Suppression
Fluorescent stain

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