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

利用CRISPR-Cas9系统制作IGFBP5a基因敲除（KO） medaka。我在drR medaka中引入了IGFBP5a基因的5个核苷酸缺失，并建立了一个异源KO系。通过异源系间的杂交，正在培育同源的IGFBP5a KO medaka系。检测了异源KO水母的耐盐性和离子细胞中转运蛋白的表达，与WT drR水母没有明显差异。我已经分别在medaka和chum salmon中定制了两种IGFBP5a抗体，但是IGFBP5a抗体并不理想，因为免疫组织化学和Western blot结果在粘液处显示非特异性信号。我建立了一种利用原位杂交链反应（ISHCR）检测转录本定位的新方法。该方法可以区分非常相似的基因异构体，并且我已经证明了na - k - atp酶(NKA) α -1a和α -1b在鲑鱼鳃中的独特定位，这是传统的使用cRNA探针的原位杂交方法无法区分的。我用同样的方法研究了IGFBP5a在鲑鱼离子细胞中的定位，初步结果表明IGFBP5a不与NKA共定位，这与斑马鱼的观察结果不同。我发现IGFBP5a在胚胎阶段的表达顺序增加，在胚胎阶段，鲑鱼会发生smoltification。然而，海水处理显著下调了鲑鱼中IGFBP5a的表达，这一观察结果与medaka不同。

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.