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Fig. 5 Generation of a floxed gata6 zebrafish allele. (A) Schematic showing insertion of loxP sites flanking exon 4 of the gata6 locus on chromosome 2. The middle line represents the gene, with rectangles as exons, and blue indicates the coding sequence, with intronic sequences not shown between the forward slashes. Gray boxes represent non-coding exon sequences. Sequences encoding the C-terminal zinc-finger DNA-binding domain are indicated in red. The upper line represents the donor oligomer including both left (LHA) and right (RHA) homology arms. Yellow triangles represent loxP sites and the black box represents the barcode. The asterisk indicates a base change to block gRNA interaction with the donor sequence. On the lower line, text represents the guide sequence with the PAM site highlighted in yellow. (B-D′) Microscopy images of 2 dpf progeny from an in-cross of gata6fl/+ adults harboring the TgBAC(ubb:loxP-TagBFP-STOP-loxP-EGFP)vcc18 allele, after injection of Cre mRNA at the one-cell stage. (B,C,D) Bright-field images show normal development in uninjected (B) and sibling embryos (D, black arrows). Injection of Cre mRNA results in pericardial edema (red arrow) in the gata6fl/fl embryo (C). (B′,C′,D′) GFP fluorescence showing successful excision of the TgBAC(ubb:loxP-TagBFP-STOP-loxP-EGFP)vcc18 reporter (C′,D′) compared with GFP-negative uninjected controls (B′). (E,E′) Representative example of genotyping 2 dpf whole embryos after Cre mRNA injection. (E) PCR screening for gata6fl/fl embryos using the 7991/7992 primer pair showing the presence (white arrowhead) or absence (black arrowhead) of the band corresponding to the wild-type gata6 allele. (E′) PCR screening for gata6fl/fl excision using the 7786/7787 primer pair showing bands corresponding to excised floxed alleles (blue arrowhead). In this particular gata6fl/fl embryo, some residual unexcised genomic DNA remains (orange arrowhead).