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

胰岛素样生长因子 5 (IGFBP5) 对鱼类渗透压调节的作用

基本信息

批准号：
22K06291
负责人：
黄國成
金额：
$ 2.66万
依托单位：
Toho University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2022
资助国家：
日本
起止时间：
2022-04-01 至 2025-03-31
项目状态：
未结题

项目摘要

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.

我使用 CRISPR-Cas9 系统制作了 IGFBP5a 敲除 (KO) 青鳉。我在 drR 青鳉的 IGFBP5a 基因中引入了 5 个核苷酸缺失，并建立了异源 KO 系。通过异源品系之间的杂交，正在培育同源IGFBP5a KO青鳉品系。对异源KO青鳉的耐盐性和离子细胞中转运蛋白的表达进行了检查，与WT drR青鳉相比没有观察到明显差异。分别在青鳉和鲑鱼中制作了两种针对 IGFBP5a 的定制抗体，但 IGFBP5a 的抗体由于免疫组化和蛋白质印迹结果并不理想结果显示粘液处存在非特异性信号。我建立了一种使用原位杂交链式反应（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亚型，并且我已经证明了鲑鱼鳃中Na-K-ATP酶（NKA）α-1a和α-1b的独特定位，这是使用cRNA探针的传统原位杂交方法无法区分的。我用同样的方法研究了鲑鱼离子细胞中IGFBP5a的定位，初步结果表明IGFBP5a不与NKA共定位，这一观察与斑马鱼不同。我发现胚胎阶段IGFBP5a表达连续增加，其中银化作用发生在鲑鱼中。然而，海水处理显着下调小鲑鱼中 IGFBP5a 的表达，这一观察结果与青鳉鱼不同。