Yamamoto Masafumi, Yamao Masafumi, Nishiyama Hiroshi, Sugihara Shinya, Nagaoka Sumiharu, Tomita Masahiro, Yoshizato Katsutoshi, Tamura Toshiki, Mori Hajime
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Biotechnol Bioeng. 2004 Dec 30;88(7):849-53. doi: 10.1002/bit.20296.
We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched G0 larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method.
我们开发了一种家蚕转基因的新方法。该方法将重组杆状病毒的使用与piggyBac转座元件的使用相结合。一种重组AcNPV，命名为辅助病毒，被设计用于在果蝇hsp70启动子的控制下表达piggyBac转座酶。另一种重组AcNPV在家蚕肌动蛋白A3启动子的控制下编码要整合到家蚕基因组中的基因，在这种情况下是绿色荧光蛋白（GFP）基因，并由piggyBac反向末端重复序列侧翼。用涂有这两种重组杆状病毒混合物的细针接种胚盘前的卵。大多数接种的幼虫孵化，并且高比例的新孵化的G0幼虫表达GFP标记。通过对G1昆虫的Southern印迹分析、对通过反向PCR分离的插入位点连接进行测序以及标记以孟德尔方式分离（如G2昆虫中绿色荧光的出现所证明）来确认转基因。因此，使用这种新方法可以轻松高效地获得转基因家蚕。
