CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line

Xue Zhang; Lihua Zhao; Runbing Jin; Min Li; Mei-Shuang Li; Rongfeng Li; Xiubin Liang

doi:10.3389/fgene.2021.664799

CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line

Front Genet. 2021 Apr 1:12:664799. doi: 10.3389/fgene.2021.664799. eCollection 2021.

Authors

Xue Zhang¹, Lihua Zhao², Runbing Jin¹, Min Li¹, Mei-Shuang Li², Rongfeng Li², Xiubin Liang^{1

3}

Affiliations

¹ Department of Pathophysiology, Nanjing Medical University, Nanjing, China.
² Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China.
³ Department of Nephrology, The Affiliated Sir Run Run Hospital of Nanjing Medical University, Nanjing, China.

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.

Keywords: CRISPR/Cas9; MIN6 cells; gene knockout; pancreatic β-cells; α-ENaC.