Dataset Information

Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss).

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 (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.

SUBMITTER: Cleveland BM

PROVIDER: S-EPMC6207780 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss).

Cleveland Beth M BM Yamaguchi Ginnosuke G Radler Lisa M LM Shimizu Munetaka M

Scientific reports 20181030 1

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 betwe ...[more]

PMID: 30375441

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Project description:BackgroundGenetic maps characterizing the inheritance patterns of traits and markers have been developed for a wide range of species and used to study questions in biomedicine, agriculture, ecology and evolutionary biology. The status of rainbow trout genetic maps has progressed significantly over the last decade due to interest in this species in aquaculture and sport fisheries, and as a model research organism for studies related to carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. We constructed a second generation genetic map for rainbow trout using microsatellite markers to facilitate the identification of quantitative trait loci for traits affecting aquaculture production efficiency and the extraction of comparative information from the genome sequences of model fish species.ResultsA genetic map ordering 1124 microsatellite loci spanning a sex-averaged distance of 2927.10 cM (Kosambi) and having 2.6 cM resolution was constructed by genotyping 10 parents and 150 offspring from the National Center for Cool and Cold Water Aquaculture (NCCCWA) reference family mapping panel. Microsatellite markers, representing pairs of loci resulting from an evolutionarily recent whole genome duplication event, identified 180 duplicated regions within the rainbow trout genome. Microsatellites associated with genes through expressed sequence tags or bacterial artificial chromosomes produced comparative assignments with tetraodon, zebrafish, fugu, and medaka resulting in assignments of homology for 199 loci.ConclusionThe second generation NCCCWA genetic map provides an increased microsatellite marker density and quantifies differences in recombination rate between the sexes in outbred populations. It has the potential to integrate with cytogenetic and other physical maps, identifying paralogous regions of the rainbow trout genome arising from the evolutionarily recent genome duplication event, and anchoring a comparative map with the zebrafish, medaka, tetraodon, and fugu genomes. This resource will facilitate the identification of genes affecting traits of interest through fine mapping and positional cloning of candidate genes.

Project description:BackgroundMaternal transcripts are accumulated in the oocyte during oogenesis to provide for protein synthesis from oocyte maturation through early embryonic development, when nuclear transcription is silenced. The maternal mRNAs have short poly(A) tails after undergoing post-transcriptional processing necessary for stabilizing them for storage. The transcripts undergo cytoplasmic polyadenylation when they are to be translated. Transcriptome analyses comparing total mRNA and elongated poly(A) mRNA content among eggs of different quality can provide insight into molecular mechanisms affecting egg developmental competence in rainbow trout. The present study used RNA-seq to compare transcriptomes of unfertilized eggs of rainbow trout females yielding different eyeing rates, following rRNA removal and poly(A) retention for construction of the libraries.ResultsThe percentage of embryos to reach the 32-cell stage at 24 h post fertilization was significantly correlated to family eyeing rate, indicating that inviable embryos were developmentally compromised before zygotic genome activation. RNA sequencing identified 2 differentially expressed transcripts (DETs) from total mRNA sequencing comparing females with low-quality (< 5% eyeing), medium-quality (30-50% eyeing), and high-quality (> 80% eyeing) eggs. In contrast, RNA sequencing from poly(A) captured transcripts identified 945 DETs between low- and high-quality eggs, 1012 between low- and medium-quality eggs, and only 2 between medium- and high-quality eggs. The transcripts of mitochondrial genes were enriched with polyadenylated transcript sequencing and they were significantly reduced in low-quality eggs. Similarly, mitochondrial DNA was reduced in low-quality eggs compared with medium- and high-quality eggs. The functional gene analysis classified the 945 DETs between low- and high-quality eggs into 31 functional modules, many of which were related to ribosomal and mitochondrial functions. Other modules involved transcription, translation, cell division, apoptosis, and immune responses.ConclusionsOur results indicate that differences in egg quality may be derived from differences in maternal nuclear transcript activation and cytoplasmic polyadenylation before ovulation, as opposed to accumulation and storage of maternal nuclear transcripts during oogenesis. Transcriptome comparisons suggest low-quality eggs suffered from impaired oxidative phosphorylation and translation. The DETs identified in this study provide insight into developmental competence in rainbow trout eggs.

Project description:BackgroundMicroRNAs (miRNAs) are small regulatory molecules which post-transcriptionally regulate mRNA stability and translation. Several microRNAs have received attention due to their role as key metabolic regulators. In spite of the high evolutionary conservation of several miRNAs, the role of miRNAs in lower taxa of vertebrates has not been studied with regard to metabolism. The liver-specific and highly abundant miRNA-122 is one of the most widely studied miRNA in mammals, where it has been implicated in the control of hepatic lipid metabolism. Following our identification of acute postprandial, nutritional and endocrine regulation of hepatic miRNA-122 isomiRNA expression in rainbow trout, we used complementary in silico and in vivo approaches to study the role of miRNA-122 in rainbow trout metabolism. We hypothesized that the role of miRNA-122 in regulating lipid metabolism in rainbow trout is conserved to that in mammals and that modulation of miRNA-122 function would result in altered lipid homeostasis and secondarily altered glucose homeostasis, since lipogenesis has been suggested to act as glucose sink in trout.ResultsOur results show that miRNA-122 was functionally inhibited in vivo in the liver. Postprandial glucose concentrations increased significantly in rainbow trout injected with a miRNA-122 inhibitor, and this effect correlated with decreases in hepatic FAS protein abundance, indicative of altered lipogenic potential. Additionally, miRNA-122 inhibition resulted in a 20% decrease in plasma cholesterol concentration, an effect associated with increased expression of genes involved in cholesterol degradation and excretion.ConclusionsOverall evidence suggests that miRNA-122 may have evolved in early vertebrates to support liver-specific metabolic functions. Nevertheless, our data also indicate that metabolic consequences of miRNA-122 inhibition may differ quantitatively between vertebrate species and that distinct direct molecular targets of miRNA-122 may mediate metabolic effects between vertebrate species, indicating that miRNA-122 - mRNA target relationships may have undergone species-specific evolutionary changes.

Dataset Information

Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss).

Publications

Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss).

Similar Datasets

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