title: Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm].
abstract: We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G2-G6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5' upstream region.
authors.affiliation: Department of Entomology and Program in Genetics and Cell and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: V
authors.initials: V
authors.lastname: Kokoza
authors.forename: A
authors.initials: A
authors.lastname: Ahmed
authors.forename: E A
authors.initials: EA
authors.lastname: Wimmer
authors.forename: A S
authors.initials: AS
authors.lastname: Raikhel
mesh_terms: D000330:Aedes; D000818:Animals; D016367:Baculoviridae; D004251:DNA Transposable Elements; D023082:Defensins; D005260:Female; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D019476:Insect Proteins / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic; D019076:Transgenes; D015004:Yellow Fever
chemical_list: D004251:DNA Transposable Elements; D023082:Defensins; D019476:Insect Proteins; D008164:Luminescent Proteins; C058181:insect defensin A; D049452:Green Fluorescent Proteins