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Fig. S3
Generation of Tg(igfbp5a:GFP) transgenic fish using Tol2-mediated BAC transgenesis. a) Schematic diagram of the BAC(igfbp5a:GFP) construct engineered. Filled boxes indicate igfbp5a ORF and open boxes indicate UTR. The iTol2 cassette and GFP reporter gene cassette were introduced into DKEYP-56B7 by homologous recombination. The igfbp5a sequence from the start codon to the end of first exon was replaced by the GFP cassette. b) The indicated DNA was subjected to Sal I digestion. Arrows showed the different bands after recombineering. c) PCR validation of the GFP cassette insertion. Forward and reverse primers were designed to target the 5'UTR of igfbp5a and the GFP gene protein coding sequence (upper panel). PCR result is shown in the lower panel. d) Representative view of GFP expression in BAC(igfbp5a:GFP)-injected F0 larvae at 120 hpf.