Germline transformation of the sawfly, Athalia rosae (Hymenoptera: Symphyta), mediated by a piggyBac-derived vector.
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.
Sumitani
M
M
Division of Bioscience, Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan.
Yamamoto
D S
DS
Oishi
K
K
Lee
J M
JM
Hatakeyama
M
M
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D003433:Crosses, Genetic; D017931:DNA Primers; D005260:Female; D005822:Genetic Vectors; D018095:Germ-Line Mutation; D049452:Green Fluorescent Proteins; D006238:Haploidy; D006926:Hymenolepis / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D012727:Sex Characteristics
D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins