Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm]

We report efficient germ-line transformation in the yellow fever mosquito A edes aegypti accomplished using the piggyBac transposable element vector pB ac[3xP3-EGFP afm]. Two,transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expres sion of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse P CR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each trans genic line. For each line, genetic analysis confirmed stability and integri ty of the entire transposon construct in the mosquito genome through the G( 2)-G(6) generations. Successful establishment of homozygous transgenic line s indicated that in both cases a non-lethal integration of the transposon i nto the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mo squitoes with different genetic backgrounds. In white-eyed transgenic mosqu itoes, the strong eye-specific expression of GFP was observed throughout al l stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-ey ed mosquitoes that were generated by crossing the 3xP3-EGFP transformants w ith the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transf ormation of wild type mosquitoes, was demonstrated by strong eye-specific G FP expression in larval and pupal stages of black-eyed hybrids of the 3xP3- EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Fin ally, analysis of the Vg-DefA transgene expression in transformants from tw o established lines demonstrated strong blood-meal activation and fat-body- specific expression regulated by the Vg 1.8-kb 5' upstream region. (C) 2001 Elsevier Science Ltd. All rights reserved.