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Abstract
A piggyBac construct carrying two green fluorescent protein (GFP)-coding sequences
one driven by Bombyx mori actin gene promoter and the other by Drosophila melanogaster
heat-shock protein 70 (hsp70) promoter were injected together with a nonautonomous
helper plasmid containing an active piggyBac transposase gene into the posterior end
of mature unfertilized eggs dissected from the ovaries of Athalia rosae (Hymenoptera:
Symphyta). These injected eggs, which developed as haploid male embryos upon artificial
activation, were cultured to adulthood. Of 278 injected eggs, 61 grew to G(0) haploid
adult males. These G(0) haploid males were individually mated to diploid females.
The progeny embryos (G(1) generation) were examined for GFP expression. Four GFP-positive
embryos (from three independent G(0) matings) were obtained. Two eclosed as diploid
adult G(1) females. Mature unfertilized eggs dissected from the GFP-positive G(1)
diploid females were activated artificially, and the resultant embryos were examined
for GFP expression, separated and cultured to adulthood (G(2) generation). The G(2)
haploid embryos segregated to GFP-positive and -negative individuals. By mating the
G(2) adult haploid males individually to diploid females, stocks were established
in which the piggyBac construct was stably integrated into the genome, as evidenced
by GFP expression and Southern blot hybridization. The piggyBac transposition occurred
at its canonical target TTAA sequence. These results, which demonstrate the first
successful stable transposon-mediated germline transformation in Hymenoptera, will
expand the usefulness of the piggyBac vector.