Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhang, Xue, Zhao, Lihua, Jin, Runbing, Li, Min, Li, Mei-Shuang, Li, Rongfeng, Liang, Xiubin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
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
_version_ 1783678999740809216
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
work_keys_str_mv AT zhangxue crisprcas9mediatedaenacknockoutinamurinepancreaticbcellline
AT zhaolihua crisprcas9mediatedaenacknockoutinamurinepancreaticbcellline
AT jinrunbing crisprcas9mediatedaenacknockoutinamurinepancreaticbcellline
AT limin crisprcas9mediatedaenacknockoutinamurinepancreaticbcellline
AT limeishuang crisprcas9mediatedaenacknockoutinamurinepancreaticbcellline
AT lirongfeng crisprcas9mediatedaenacknockoutinamurinepancreaticbcellline
AT liangxiubin crisprcas9mediatedaenacknockoutinamurinepancreaticbcellline