Gal4-based enhancer-trapping in the malaria mosquito Anopheles stephensi.

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TitleGal4-based enhancer-trapping in the malaria mosquito Anopheles stephensi.
Publication TypeJournal Article
Year of Publication2012
AuthorsO'Brochta, DA, Pilitt, KL, Harrell, RA, Aluvihare, C, Alford, RT
JournalG3 (Bethesda)
Volume2
Issue11
Pagination1305-15
Date Published2012 Nov
ISSN2160-1836
KeywordsAnimals, Animals, Genetically Modified, Anopheles, Base Sequence, DNA Transposable Elements, Drosophila, Enhancer Elements, Genetic, Gene Expression Regulation, Genes, Reporter, Green Fluorescent Proteins, HSP70 Heat-Shock Proteins, Molecular Sequence Data, Promoter Regions, Genetic, Protein Binding, Recombinant Fusion Proteins, Transcription Factors, Transposases
Abstract

Transposon-based forward and reverse genetic technologies will contribute greatly to ongoing efforts to study mosquito functional genomics. A piggyBac transposon-based enhancer-trap system was developed that functions efficiently in the human malaria vector, Anopheles stephensi. The system consists of six transgenic lines of Anopheles stephensi, each with a single piggyBac-Gal4 element in a unique genomic location; six lines with a single piggyBac-UAStdTomato element; and two lines, each with a single Minos element containing the piggyBac-transposase gene under the regulatory control of the hsp70 promoter from Drosophila melanogaster. Enhancer detection depended upon the efficient remobilization of piggyBac-Gal4 transposons, which contain the yeast transcription factor gene Gal4 under the regulatory control of a basal promoter. Gal4 expression was detected through the expression of the fluorescent protein gene tdTomato under the regulatory control of a promoter with Gal4-binding UAS elements. From five genetic screens for larval- and adult-specific enhancers, 314 progeny were recovered from 24,250 total progeny (1.3%) with unique patterns of tdTomato expression arising from the influence of an enhancer. The frequency of piggyBac remobilization and enhancer detection was 2.5- to 3-fold higher in female germ lines compared with male germ lines. A small collection of enhancer-trap lines are described in which Gal4 expression occurred in adult female salivary glands, midgut, and fat body, either singly or in combination. These three tissues play critical roles during the infection of Anopheles stephensi by malaria-causing Plasmodium parasites. This system and the lines generated using it will be valuable resources to ongoing mosquito functional genomics efforts.

DOI10.1534/g3.112.003582
Alternate JournalG3 (Bethesda)
PubMed ID23173082
PubMed Central IDPMC3484661
Grant ListR01 A10 70812 / / PHS HHS / United States
R44 AI0-77262 / AI / NIAID NIH HHS / United States