Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout oncorhynchus mykiss

Authors: Cleveland, Beth; YAMAGUCHI, GINNOSUKE - Hokkaido University; Radler, Lisa; SHIMIZU, MUNETAKA - Hokkaido University

Submitted to: Aquaculture America Conference
Publication Type: Abstract Only
Publication Acceptance Date: 8/16/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: 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 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, 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.