Expression of foreign Abcc4 in transgenic zebrafish. (A) A primer pair (Flag-F and ABC-R3) was designed to recognize the abcc4 coding sequence (CDS) and Flag sequence. (B) Positive F2 fish were individually crossed with WT fish to obtain positive F3 embryos. Sixty larvae at 96 hpf were pooled for RNA extraction and subsequent RT-PCR analysis with a primer pair (Flag-F and ABC-R3). WT: Wild-type zebrafish embryos at the same developmental stage. F3 abcc4-1# and F3 abcc4-2# indicated the pooled larval zebrafish at 96 hpf from two positive F2 zebrafish, which were individually crossed with WT fish, respectively. (C) qPCR analysis for abcc4 mRNA levels with a primer pair (abcc4–qPCR-F and abcc4–qPCR-R). (D) Western blotting analysis of Flag-tagged Abcc4 expression in F3 embryos. (E) WISH analysis of abcc4 expression in WT and transgenic zebrafish larvae. Arrow heads indicate the brain and intestine.