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CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line
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 t...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047203/ https://www.ncbi.nlm.nih.gov/pubmed/33868391 http://dx.doi.org/10.3389/fgene.2021.664799 |
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author | Zhang, Xue Zhao, Lihua Jin, Runbing Li, Min Li, Mei-Shuang Li, Rongfeng Liang, Xiubin |
author_facet | Zhang, Xue Zhao, Lihua Jin, Runbing Li, Min Li, Mei-Shuang Li, Rongfeng Liang, Xiubin |
author_sort | Zhang, Xue |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8047203 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-80472032021-04-16 CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line Zhang, Xue Zhao, Lihua Jin, Runbing Li, Min Li, Mei-Shuang Li, Rongfeng Liang, Xiubin Front Genet Genetics 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. Frontiers Media S.A. 2021-04-01 /pmc/articles/PMC8047203/ /pubmed/33868391 http://dx.doi.org/10.3389/fgene.2021.664799 Text en Copyright © 2021 Zhang, Zhao, Jin, Li, Li, Li and Liang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Genetics Zhang, Xue Zhao, Lihua Jin, Runbing Li, Min Li, Mei-Shuang Li, Rongfeng Liang, Xiubin CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line |
title | CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line |
title_full | CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line |
title_fullStr | CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line |
title_full_unstemmed | CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line |
title_short | CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line |
title_sort | crispr/cas9-mediated α-enac knockout in a murine pancreatic β-cell line |
topic | Genetics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047203/ https://www.ncbi.nlm.nih.gov/pubmed/33868391 http://dx.doi.org/10.3389/fgene.2021.664799 |
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