Figure 1.
Strategy for FLP recombinase-mediated site-specific recombination in silkworms.
The piggyBac-derived vector pBac{3×P3-DsRed; FRT-3×P3-EGFP-SV40-FRT} (A) was inserted into the TTAA site of the G0 silkworm germ cell genome to produce a stable G1 transgenic target strain (TTS) (C) mediated by piggyBac transposase derived from plasmid pHA3PIG (B). The TTS was transgenic for a 3×P3 promoter-driving DsRed gene (red box) expression cassette and a cassette that was flanked by two 48-bp FRT sites (black triangles) in the same orientation. A 3×P3 promoter-driving EGFP gene (green box) was placed internally to the two FRT sites. Site-specific recombination between the two FRT sites of G2 TTS germ cell genome (E), mediated by FLP recombinase derived from helper vector pSLA3-FLP (D), result in the deletion of the 3×P3-EGFP expression cassette from the genome of G3-positive site-specific recombination strain (SSRS) individuals (F). 3×P3, 3×P3 promoter; SV40, SV40 polyadenylation signal sequence; A3, silkworm cytoplasmic actin 3 promoter; A3 polyA, polyadenylation signal sequence of silkworm A3 gene; pBacL, left arm of piggyBac transposon; pBacR, right arm of piggyBac transposon. The restriction enzyme sites for the construction of recombinant vectors are shown.
Table 1.
Injection of piggyBac-derived vectors in G0 silkworm embryos of the strain Dazao.
Figure 2.
Expression of the DsRed and EGFP genes in TTS silkworms.
(A–C) show white light (A), RFP-fluorescent (B) and GFP-fluorescent (C) images of 6-day-old G1 TTS-1 embryos. Arrowheads denote the position of the RFP and GFP fluorescence. (D–F) show white light (D), RFP-fluorescent (E) and GFP-fluorescent (F) images of the G1 TTS-1 adults.
Table 2.
Identification of the genomic insertion sites of the pBac{3×P3-DsRed; FRT-3×P3-EGFP-SV40-FRT} vector by inverse PCR.
Table 3.
Injection of FLP recombinase expression vector into silkworm embryos obtained by crossing heterozygous G1 TTSs males with wild-type females.
Figure 3.
Expression of the DsRed and EGFP genes detected at different developmental stages of G3 silkworm individuals.
(A–C) show white light (A), RFP-fluorescent (B) and GFP-fluorescent (C) images of 6-day-old G3 silkworm embryos. (D–F) show white light (D), RFP-fluorescent (E) and GFP-fluorescent (F) images of the 7-day-old G3 silkworm embryos. The DsRed- and GFP-positive non-site-specific recombinant transgenic embryos are highlighted with an arrowhead; DsRed-positive site-specific recombinant transgenic embryos are highlighted with a triangle, and wild-type embryos are indicated with an asterisk.
Figure 4.
Expression of the DsRed and EGFP genes in larvae and adults from TTS and SSRS silkworms.
(A) The newly hatched larvae of wild-type strain (a–c), TTS-1 (d–f) and SSRS-1 (g–i) showing white light (a,d,g), RFP fluorescence (b,e,h) and GFP fluorescence (c,f,i) in the developing larval ocelli. (B) The adults of the wild-type strain (a–c), TTS-1 (d–f) and SSRS-1 (g–i) showing white light (a,d,g), RFP fluorescence (b,e,h) and GFP fluorescence (c,f,i) in the compound eye.
Figure 5.
Molecular confirmation of FLP-mediated site-specific recombination in silkworm.
(A) Schematic map of the FRT-3×P3-EGFP-SV40-FRT cassette before (top) and after (bottom) site-specific excision in transgenic silkworms. Two FRT sites (black triangles) before (top) and after (bottom) recombination are flanked by different restriction sites. The SpeI, XhoI and BglII are shown, as are the recognition sites of P–F/P–R primer pair used for PCR analysis. A 1514-bp amplicon was detected from the G1 TTS genome (top) and a 183-bp amplicon was detected from the G3 SSRS genome (bottom). XhoI- and BglII-digested genomic DNAs were hybridized to DsRed and EGFP probes. The EGFP probe hybridized fragment size calculated before recombination (top) was 1331 bp. HaeIII-digested genomic DNAs were used to make templates for inverse PCR. PLF/PLR and PRF/PRR are the piggyBac left and right arm (pBacL and pBacR) primer pairs, respectively. (B) PCR confirmation of FLP recombinase-mediated site-specific excision in G3 transgenic silkworms. Genomic DNAs from two adults of G1 TTSs and eight adults of G3 SSRSs were used as DNA templates for PCR to confirm excision of the FRT-flanked 3×P3-EGFP expression cassette with primers P–F/P–R. Wild-type silkworm (W) were used as a control. Lane M, the Trans2K Plus DNA Marker. (C) Sequencing result of the 183-bp PCR products from all SSRS-positive individuals. Horizontal arrows show the primer pair P–F/P–R. Black background sequence shows a 48-bp recombinant FRT site in the genomic DNA. (D) Southern blotting analysis of FLP recombinase-mediated site-specific recombination. 25 ug genomic DNA samples were digested with XhoI and BglII, separated by agarose gel electrophoresis, and hybridized with DsRed and EGFP-specific probes. The individual DNA hybridization patterns of the wild-type (W), TTS-1 (G1–3), SSRS-1 (G3–5, G3–7), TTS-2 (G1–2) and SSRS-2 (G3–10, G3–14) lanes are shown. Red triangles and green triangles denote the signals for the DsRed and EGFP probes, respectively.
Table 4.
Comparison of the recombination efficiency mediated by the FLP/FRT system in other higher eukaryotes.