title: Gal4-based enhancer-trapping in the malaria mosquito Anopheles stephensi.
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.
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, MD 20850, USA. dobrocht@umd.edu
authors.forename: David A
authors.initials: DA
authors.lastname: O'Brochta
authors.forename: Kristina L
authors.initials: KL
authors.lastname: Pilitt
authors.forename: Robert A
authors.initials: RA
authors.lastname: Harrell
authors.forename: Channa
authors.initials: C
authors.lastname: Aluvihare
authors.forename: Robert T
authors.initials: RT
authors.lastname: Alford
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000852:Anopheles / Q000235:genetics*; D001483:Base Sequence; D004251:DNA Transposable Elements; D004330:Drosophila / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C473499:Minos transposase; D019895:Transposases