New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements.
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.
Yamamoto
Masafumi
M
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Yamao
Masafumi
M
Nishiyama
Hiroshi
H
Sugihara
Shinya
S
Nagaoka
Sumiharu
S
Tomita
Masahiro
M
Yoshizato
Katsutoshi
K
Tamura
Toshiki
T
Mori
Hajime
H
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx / Q000196:embryology / Q000235:genetics* / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods*; D019895:Transposases / Q000235:genetics / Q000378:metabolism
D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D019895:Transposases