An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.
We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs.
State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, People's Republic of China.
Dingpei
D
Long
State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, People's Republic of China.
Weijian
W
Lu
Guangxi Research Academy of Seficultural Science, Guangxi Nanning 530007, People's Republic of China.
Yuli
Y
Zhang
Guangxi Research Academy of Seficultural Science, Guangxi Nanning 530007, People's Republic of China.
Lihui
L
Bi
State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, People's Republic of China.
Zhonghuai
Z
Xiang
State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, People's Republic of China.
Aichun
A
Zhao
D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism; D003433:Crosses, Genetic; D004251:DNA Transposable Elements; D005260:Female; D015870:Gene Expression; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D042822:Genomic Instability; D059765:Homologous Recombination; D008297:Male; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D019076:Transgenes
D004251:DNA Transposable Elements