Biology subjects

Datsomor, A. K.

Publications and source records attributed to Datsomor, A. K..

3 recordsLinked to original sources

bioRxiv2022

Functional validation of transposable element derived cis-regulatory elements in Atlantic salmon

BackgroundTransposable elements (TEs) are hypothesized to play important roles in shaping genome evolution following whole genome duplications (WGD), including rewiring of gene regulation. In a recent analysis, duplicate gene copies that had evolved higher expression in liver following the salmonid WGD ~100 million years ago were associated with higher numbers of predicted TE-derived cis-regulatory elements (TE-CREs). Yet, the ability of these TE-CREs to recruit transcription factors (TFs) in vivo and impact gene expression remains unknown. ResultsHere, we evaluated the gene regulatory functions of 11 TEs using luciferase promoter reporter assays in Atlantic salmon (Salmo salar) primary liver cells. Canonical Tc1-Mariner elements from intronic regions showed no or small repressive effects on transcription. However, other TE-derived cis-regulatory elements upstream of transcriptional start sites increased expression significantly. ConclusionOur results question the hypothesis that TEs in the Tc1-Mariner superfamily, which were extremely active following WGD in salmonids, had a major impact on regulatory rewiring of gene duplicates, but highlights the potential of other TEs in post-WGD rewiring of gene regulation in the Atlantic salmon genome.

evolutionary biology↗

bioRxiv2022

Efficient transfection of Atlantic salmon primary hepatocyte cells for functional assays and gene editing

The expansion of genomic resources for Atlantic salmon over the past half decade has enabled efficient interrogation of genetic traits by large-scale correlation of genotype to phenotype. Moving from correlation to causation will require genotype-phenotype relationships to be tested experimentally in a cost-efficient and cell context relevant manner. To enable such future experiments, we have developed a method for the isolation and genetic manipulation of primary hepatocytes from Atlantic salmon for use in heterologous expression, reporter assay, and gene editing experiments. We chose the liver as the tissue of interest because it is the metabolic hub and many current Atlantic salmon research projects focus on understanding metabolic processes to improve traits such as growth rate, total fat content, and omega-3 content. We find that isolated primary hepatocytes are optimally transfected with both plasmid and ribonucleoprotein using a Neon electroporator at 1400 V, 10 ms, and 2 pulses. Transfection efficiency with plasmid and cutting efficiency with ribonucleoprotein was optimally 46% and 60%, respectively. We also demonstrate a 26-fold increase in luciferase expression under the promoter of the key liver metabolic gene, elovl5b, compared to empty vector, in line with expected liver-specific expression. Taken together, this work provides a valuable resource enabling transfection and gene editing experiments in a context relevant and cost-effective system.

molecular biology↗

bioRxiv2020

Targeted mutagenesis of {triangleup}5 and {triangleup}6 fatty acyl desaturases induce multiplex-mutagenesis and lipogenesis in Atlantic salmon (Salmo salar)

With declining wild fish populations, farmed Atlantic salmon (Salmo salar) has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA) including 20:5n-3 and 22:6n-3. However, the introduction of plant-based oil in fish diets has reduced the content of these beneficial LC-HUFA. The capability of biosynthesis of LC-HUFAs depends on fatty acids supplied in diets and the genetic potential residing in the fish. Key proteins involved in LC-HUFA synthesis in salmon include fatty acid desaturases 2 (Fads2). In a recent study we used CRISPR/Cas9 to generate two F0 mutant strains of salmon, 1){Delta} 6abc/5Mt with mutations in{Delta} 5fads2, {Delta}6fads2-a, {Delta}6fads2-b and {Delta}6fads2-c genes, and 2){Delta} 6bcMt with mutations in{Delta} 6fads2-b and {Delta}6fads2-c genes. The CRISPR mutated salmon (crispants) had reduced levels of LC-HUFA and expression of targeted fads2 genes. In present study we apply whole transcriptome analysis on these fads2 crispants. Our purpose is to evaluate the genetic mosaicism in fads2 crispants and the effect these mutations had on other lipid metabolism pathways in fish. Both{Delta} 6abc/5Mt and{Delta} 6bcMt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. Skipping of a CRISPR-targeted exon was observed in{Delta} 6fads2-a gene of{Delta} 6abc/5Mt salmon. The{Delta} 6abc/5Mt fish also displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation of srebp1 genes as well as genes involved in fatty acid de-novo synthesis, fatty acid {beta}-oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of{Delta} 6bcMt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability.

molecular biology↗