Abstract
Many ion channels participate in controlling insulin synthesis and secretion of pancreatic β-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic β-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout α-ENaC gene in a murine pancreatic β-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of α-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the α-ENaC gene-knockout (α-ENaC−/−) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in α-ENaC−/− MIN6 cells. Therefore, α-ENaC−/− MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of α-ENaC in pancreatic β-cells.
Highlights
Diabetes mellitus (DM) is usually characterized by an insulin deficiency due to pancreatic β-cell dysfunction including insulin secretion and insulin action, and disorder of insulin secretion promotes diabetes progression (Roden, 2016; Chatterjee et al, 2017)
In α subunit of ENaC (α-ENaC)−/− MIN6 cell line, the top 10 off-target sites (OTSs) for each single-guide RNA (sgRNA) were selected in Supplementary Table S1, and the genomic regions covering the OTSs were PCR amplified using the primers listed in Supplementary Table S2
The T7E1 enzyme digestion results showed that Cas9-sgRNA1 and Cas9-sgRNA3 targeting on the α-ENaC gene were highly efficient (Figure 1B), and the α-ENaC Knockout in MIN6 cells sequence analysis showed their mutation efficiencies were 18.7 and 11.2%, respectively (Supplementary Figure S1)
Summary
Diabetes mellitus (DM) is usually characterized by an insulin deficiency due to pancreatic β-cell dysfunction including insulin secretion and insulin action, and disorder of insulin secretion promotes diabetes progression (Roden, 2016; Chatterjee et al, 2017). ENaC is mainly distributed in the distal convoluted tubules and collecting ducts of the distal nephrons, and acts as a rate-limiting effect on sodium reabsorption by mediating the transmembrane transport of sodium ions. This plays an important role in maintaining sodium’s own balance, extracellular fluid volume and stabilizing blood pressure (Rossier, 2014; Hanukoglu and Hanukoglu, 2016). The derived α-ENaC deficient MIN6 cell line will become an effective tool for elucidating the α-ENaC function and its molecular mechanism in diabetes mellitus
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