Sumitani M, Yamamoto D S, Oishi K, Lee J M, Hatakeyama M
Division of Bioscience, Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan.
Insect Biochem Mol Biol. 2003 Apr;33(4):449-58. doi: 10.1016/s0965-1748(03)00009-2.
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.
将携带两个绿色荧光蛋白（GFP）编码序列的piggyBac构建体（一个由家蚕肌动蛋白基因启动子驱动，另一个由黑腹果蝇热休克蛋白70（hsp70）启动子驱动）与含有活性piggyBac转座酶基因的非自主辅助质粒一起注射到从玫瑰三节叶蜂（膜翅目：叶蜂科）卵巢中解剖出的成熟未受精卵的后端。这些注射后的卵在人工激活后发育为单倍体雄性胚胎，并培养至成年。在278个注射的卵中，有61个发育为G(0)单倍体成年雄性。这些G(0)单倍体雄性分别与二倍体雌性交配。检查后代胚胎（G(1)代）的GFP表达情况。获得了四个GFP阳性胚胎（来自三个独立的G(0)交配）。其中两个羽化成为二倍体成年G(1)雌性。从GFP阳性的G(1)二倍体雌性中解剖出成熟未受精卵进行人工激活，并检查所得胚胎的GFP表达情况，将其分离并培养至成年（G(2)代）。G(2)单倍体胚胎分离为GFP阳性和阴性个体。通过将G(2)成年单倍体雄性分别与二倍体雌性交配，建立了piggyBac构建体稳定整合到基因组中的品系，GFP表达和Southern印迹杂交证明了这一点。piggyBac转座发生在其典型的靶序列TTAA处。这些结果首次证明了在膜翅目中成功实现稳定的转座子介导的种系转化，将扩大piggyBac载体的应用范围。
