Figure 1

Strategy for the selection of high-efficiency transgene expression and the post-integration stabilization of the transgene using PB-TP vector in silkworms.

The initial transformation uses the PB-TP vector (A) carrying a composite transposon comprising two pairs of opposed transposon arms. The PB-TP structurally combines four different transposons (R1–L1, R1–L2, R2–L1 and L2–R2). The desired transposon R1–L1 was inserted into the TTAA site of the G0 silkworm germ cell genome to produce the TS1-RgG individuals (C) mediated by piggyBac transposase derived from plasmid pHA3PIG (B). TS1-RgG individuals containing a single copy of the R1–L1 construct in their genomes (selected by the 3×P3-DsRed, FibH-EGFP and 3×P3-EGFP markers) and expressing high-level EGFP in their cocoons were identified. The flanking transposons (R1–L2 and R2–L1) can then be eliminated in TS2-RgG germ-cell genome (D) by reexposure to transposase, mediated by heat shock treatment, resulting in the removal of one flanking transposon (R2–L1 or R1–L2) or both flanking transposons from the TS3-Rg, TS3-gG, or TS3-g genome (E–G). TS3-gG individuals in which only the R1–L2 transposon was deleted from the genome (F) were also used for a secend round of excision, completely eliminating the transposons (as above), leaving only the FibH-EGFP expression cassette flanked by two 39-bp attP sites (triangles with diagonal lines) in the same orientation in the TS4-g genome (H). 3×P3, 3×P3 promoter; DsRed, DsRed gene (red box); SV40, SV40 polyadenylation signal sequence; FibH, 5′-flanking sequence of the silkworm fibroin heavy chain gene (~2.3-kb); EGFP, EGFP gene (green box); LBS, light chain binding site sequence of the silkworm fibroin heavy chain gene; Hsp70, D. Melanogaster hsp70 promoter; A3, silkworm cytoplasmic actin 3 promoter; PBase, piggyBac transposase gene (blue box).