Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss).
Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss).
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DOI：
10.1038/s41598-018-34326-6
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发表时间：
2018-10-30
影响因子：
4.6
通讯作者：
Shimizu M
中科院分区：
文献类型：
--
作者：
Cleveland BM;Yamaguchi G;Radler LM;Shimizu M
在鲑鱼中，大多数循环中的胰岛素样生长因子- i （IGF- i）与IGF结合蛋白（IGFBP）结合，其中IGFBP-2b在循环中含量最多。我们使用CRISPR/Cas9方法通过共同靶向基因编辑IGFBP-2b1和IGFBP-2b2亚型（代表鲑鱼特异性基因重复）来破坏功能性IGFBP-2b蛋白的表达。24条虹鳟鱼携带IGFBP-2b1和IGFBP-2b2基因突变。突变鱼对IGFBP-2b1和IGFBP-2b2基因的破坏率分别为8-100%和2-83%，个体间基因突变率呈正相关（P < 0.001）。IGFBP-2b蛋白分析显示血浆IGFBP-2b丰度降低至对照水平的0.04 - 0.96倍。在孵化后8个月和10个月，突变体的血浆IGF-I、体重和叉长都减少，这支持了IGFBP-2b对携带IGF-I的重要作用。尽管突变体的血浆IGF-I和IGFBP-2b水平降低，但在孵化后10至12个月，突变体的生长迟缓程度较轻（P < 0.05），表明发生了代偿性生长反应。这些发现表明，利用CRISPR/Cas9和配体印迹技术进行基因编辑是一种表征鲑鱼中重复IGFBP基因蛋白水平功能的可行方法，有助于揭示igf相关的内分泌机制。
In salmonids, the majority of circulating insulin-like growth factor-I (IGF-I) is bound to IGF binding proteins (IGFBP), with IGFBP-2b being the most abundant in circulation. We used CRISPR/Cas9 methodology to disrupt expression of a functional IGFBP-2b protein by co-targeting for gene editing IGFBP-2b1 and IGFBP-2b2 subtypes, which represent salmonid-specific gene duplicates. Twenty-four rainbow trout were produced with mutations in the IGFBP-2b1 and IGFBP-2b2 genes. Mutant fish exhibited between 8–100% and 2–83% gene disruption for IGFBP-2b1 and IGFBP-2b2, respectively, with a positive correlation (P < 0.001) in gene mutation rate between individual fish. Analysis of IGFBP-2b protein indicated reductions in plasma IGFBP-2b abundance to between 0.04–0.96-fold of control levels. Plasma IGF-I, body weight, and fork length were reduced in mutants at 8 and 10 months post-hatch, which supports that IGFBP-2b is significant for carrying IGF-I. Despite reduced plasma IGF-I and IGFBP-2b in mutants, growth retardation in mutants was less severe between 10 and 12 months post-hatch (P < 0.05), suggesting a compensatory growth response occurred. These findings indicate that gene editing using CRISPR/Cas9 and ligand blotting is a feasible approach for characterizing protein-level functions of duplicated IGFBP genes in salmonids and is useful to unravel IGF-related endocrine mechanisms.
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