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PMID: 18549956 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

A transgenic sensor strain for monitoring the RNAi pathway in the yellow fever mosquito, Aedes aegypti.

Insect biochemistry and molecular biology ·Vol. 38 ·No. 7 ·2008-07-00 ·Pages 705-13

Adelman ZN, Anderson MA, Morazzani EM, Myles KM

Abstract

The RNA interference pathway functions as an antiviral defense in invertebrates. In order to generate a phenotypic marker which "senses" the status of the RNAi pathway in Aedes aegypti, transgenic strains were developed to express EGFP and DsRED marker genes in the eye, as well as double-stranded RNA homologous to a portion of the EGFP gene. Transgenic "sensor" mosquitoes exhibited robust eye-specific DsRED expression with little EGFP, indicating RNAi-based silencing. Cloning and high-throughput sequencing of small RNAs confirmed that the inverted-repeat transgene was successfully processed into short-interfering RNAs by the mosquito RNAi pathway. When the A. aegypti homologues of the genes DCR-2 or AGO-2 were knocked down, a clear increase in EGFP fluorescence was observed in the mosquito eyes. Knockdown of DCR-2 was also associated with an increase in EGFP mRNA levels, as determined by Northern blot and real-time PCR. Knockdown of AGO-3, a gene involved in the germline-specific piRNA pathway, did not restore EGFP expression at either the mRNA or protein level. This transgenic sensor strain can now be used to identify other components of the mosquito RNAi pathway and has the potential to be used in the identification of arboviral suppressors of RNAi.

MeSH Terms
Aedes/genetics,metabolism Animals Animals, Genetically Modified Cloning, Molecular DNA Transposable Elements Eye/metabolism Female Gene Silencing Green Fluorescent Proteins/genetics,metabolism Humans Insect Proteins/genetics,metabolism Insect Vectors/genetics,metabolism Luminescent Proteins/genetics,metabolism Male Mutagenesis RNA Interference RNA, Small Interfering/genetics Species Specificity Yellow Fever/virology
Chemicals
DNA Transposable Elements Insect Proteins Luminescent Proteins RNA, Small Interfering enhanced green fluorescent protein fluorescent protein 583 Green Fluorescent Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Adelman Zach N
Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. zachadel@vt.edu
Anderson Michelle A E
Morazzani Elaine M
Myles Kevin M
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Article Info
Journal
Insect biochemistry and molecular biology
Abbr.
Insect Biochem Mol Biol
ISSN
0965-1748
DOI
10.1016/j.ibmb.2008.04.002
Email
zachadel@vt.edu
Published
2008-07-00
Epub
2008-00-22
Pages
705-13
Language
English
Region
England
NLM ID
9207282
PMCID
PMC2518454
Subset
IM
Grants
NIAID NIH HHS · R03 AI070198 · United States
NIAID NIH HHS · R03 AI070198-01A1 · United States
NIAID NIH HHS · 1R03 AI070198-01A1 · United States
External Links
PubMed source DOI: 10.1016/j.ibmb.2008.04.002 PMC Full Text View journal details
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