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

Abstract

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 the 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 1,400 V, 10 ms, and 2 pulses. Transfection efficiency with plasmid and cutting efficiency with ribonucleoprotein were optimally 46% and 60%, respectively. We also demonstrate a 26 times increase in luciferase expression under the promoter of the key liver metabolic gene, elovl5b, compared to an 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.

Keywords: Atlantic salmon; CRISPR; cell culture; hepatocyte; primary cells.

Fig. 1.

Schematic of the primary hepatocyte isolation procedure and electroporation using the Neon electroporation system.

Fig. 2.

Impact of electroporation programs on transfection efficiency and viability and demonstration of functional reporter assay in Atlantic salmon primary hepatocytes. a and b) The different electroporation programs assessed show varying effects on transfection of hepatocytes with program P16 (Table 1) giving highest transfection efficiency. Transfection efficiency was measured by flow cytometry. c) Evaluation of cell viability by resazurin viability assay at 48-hr post-transfection showed slight reduction in cell viability for all programs compared to non-transfected control. d) Normalized luciferase signal for Atlantic salmon elovl5b promoter compared to the empty vector.

Fig. 3.

Cutting efficiency of RNPs targeting Atlantic salmon P53 (NCBI geneID:106602901) using four electroporation programs. Sanger sequencing traces for each electroporation program are shown. The gRNA binding site is underlined in the WT trace and the cut site is indicated by a vertical dashed line in all traces. ICE deconvolutions are shown for each program. Bar plots (right) indicate total percent cutting efficiency (wide bars) and contribution of each indel (narrow bars). Indels of length 0 are unaltered WT sequence and negative indels are deletions. No insertions were detected.