Fig. 3

PhiC31 integrase mediates DNA integration via recombination between attB and the genomic attP site. (A) A diagram depicting the integration of ubi:EGFP at the attPtyr_1 locus. PhiC31 integrase mediates recombination between the plasmid DNA containing the attB sequence and the genomic attP site, resulting in single-copy integration of the entire plasmid. The integrated DNA (thick black line) is flanked by two attP/attB composite sequences named attR and attL, which can be detected via PCR using two sets of primers (F1/R1 and F2/R2) as indicated. F1 and R2 are tyr-specific, whereas R1 and F2 are plasmid-specific. (B) PCR analysis of attR and attL in individual embryos microinjected with the phiC31 mRNA and pDestattB_ubi:EGFP. Red arrows indicate the expected product sizes of attR (369 bps, top panel) and attL (534 bps, bottom panel). The bracket with an asterisk on the right side of the bottom gel indicates non-specific amplifications from the F2/R2 primer set. The embryos with the correct size products from both attR and attL PCR are indicated by red numbers on the top. (C) Sanger sequencing of the attR (top chromatogram) and attL (bottom chromatogram) PCR products demonstrates precise DNA integration. Reference sequences for attR and attL are shown in the box above the chromatograms. Sequences originating from attP and attB are indicated in brown and turquoise, respectively. The ‘AA’ crossover site is shown in red.