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

Insect Biochem Mol Biol. 2001 Nov 1;31(12):1137-43. doi: 10.1016/s0965-1748(01)00120-5.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aedes*
  • Animals
  • Baculoviridae*
  • DNA Transposable Elements
  • Defensins*
  • Female
  • Genetic Vectors*
  • Green Fluorescent Proteins
  • Insect Proteins / genetics*
  • Luminescent Proteins / genetics
  • Promoter Regions, Genetic
  • Transformation, Genetic*
  • Transgenes
  • Yellow Fever


  • DNA Transposable Elements
  • Defensins
  • Insect Proteins
  • Luminescent Proteins
  • insect defensin A
  • Green Fluorescent Proteins