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Fig. 1
Generation of attP/attB-recombinant zebrafish transgenics. A, B: cmlc2:EGFP-marked attP sites were introduced into the zebrafish genome by Tol2-mediated transgenesis. Presence of attP site allows for integration of attB-bearing transgene plasmid (shown ubi:EGFP as example) upon co-injection with phiC31 integrase mRNA into offspring from heterozygous cmlc2:EGFP-positive attP landing site carriers crossed out to wt; asterisks indicate cmlc2:EGFP-positive hearts. C: Injection of attB_ubi:EGFP together with phiC31 integrase mRNA causes mosaic fluorophore expression in the injected F0 larvae (top panel) and adult (bottom panel) of representative attP landing site lines, as detected by fluorescence microscopy; asterisk indicates cmlc2:EGFP-positive hearts. D: Agarose gel analysis of attL/attR-specific PCR on genomic DNA of 8 individual cmlc2:EGFP-positive, ubi:EGFP-mosaic embryos (48 hpf); arrows in the schematic indicate primer positioning, arrowheads highlight PCR product sizes. E: Sequencing of the verified precise attB plasmid integration into the genomic attP site. Blue boxes indicate the TTG crossover site. Sequencing of amplicons 1 through 8 from attL- and attR-specific amplicons verified precise attB plasmid integration into the genomic attP site by comparison to reference sequences (blue row). Representative sequencing graphs demonstrate sequencing results (bottom panels).