The roles of insulin-like growth factor 5 (IGFBP5) on fish osmoregulation

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I have made IGFBP5a knockout (KO) medaka using CRISPR-Cas9 system. I have introduced a 5-nucleotide deletion in the IGFBP5a gene in drR medaka and established a heterologous KO line. With crossing between the heterologous line, it is in the process to breed homologous IGFBP5a KO medaka line.The salinity tolerance and transporter expression in the ionocytes in heterologous KO medaka was examined and no apparent difference was observed compared with the WT drR medaka.I have made two custom antibodies for the IGFBP5a in medaka and chum salmon respectively, but the antibodies for IGFBP5a were not desirable as the immunohistochemistry and Western blot results showed non-specific signals at the mucus.I have established a novel method to detect the transcript localization using in situ hybridization chain reaction (ISHCR). The method can distinguish closely resemble gene isoforms and I have demonstrated the distinct localization of Na-K-ATPase (NKA) alpha-1a and alpha-1b in chum salmon gill, which cannot be distinguished by conventional in situ hybridization method using cRNA probes. I have used the same method to study the localization of IGFBP5a in chum salmon ionocytes, and initial result suggested that the IGFBP5a is not colocalized with NKA, an observation different from that of zebrafish.I found a sequential increase in IGFBP5a expression during embryonic stages, in which the smoltification occurs in chum salmon. However, seawater treatment downregulated the IGFBP5a expression significantly in smolting chum salmon, an observation that is different from that of medaka.