An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.

Dingpei Long,Lihui Bi,Weijian Lu,Yuli Zhang,Aichun Zhao,Zhonghuai Xiang

doi:10.1038/srep08802

Abstract

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.

Highlights

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 heavy chain (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
piggyBac-derived target plasmid (PB-TP) structurally combines four different transposons (R1–L1, R1–L2, R2–L1, and L2–R2) that can potentially be expressed from this type of construct, and each transposon can be identified by a different combination of the 33P3-DsRed (R), 33P3-EGFP (G), and FibH-EGFP (g) fluorescent markers (Figure 1A)

Summary

Introduction

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. PiggyBac is currently the most widely used transposon vector and has been successfully used for transgenic engineering in more than 30 insect species, including D. melanogaster[10], Ceratitis capitata[11], Tribolium castaneum[12], Bombyx mori[13], Aedes aegypti[14], and Apis mellifera[15] It facilitates the extension of enhancer-trapping strategies, allowing the identification and functional analysis of genes, the expression of foreign proteins, the biological control of pest species, and other genetic engineering operations[3]. It is almost impossible to repeatedly introduce several different exogenous genes into a specific target locus using only piggyBac or other transposons

Methods

Results

Conclusion