Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9
CRISPR-Cas9 gene editing has made it possible to specifically edit genes in a myriad of target cells. Here, a method for isoform-specific editing and clonal selection in Madin-Darby canine kidney (MDCK) epithelial cells is described in detail. This approach was used to address a long-standing questi...
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doaj-16779e2147184e8b8545c9d2bbd355cc2021-01-02T05:11:14ZengElsevierMethodsX2215-01612020-01-017101149Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9James M. Readler0Amal S. AlKahlout1Abimbola O. Kolawole2Katherine J.D.A. Excoffon3Biomedical Sciences PhD Program, Wright State University, Dayton, OH 45435, United States; Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Hwy, 235a BS, Dayton, OH 45435, United States; Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, United StatesDepartment of Biological Sciences, Wright State University, 3640 Colonel Glenn Hwy, 235a BS, Dayton, OH 45435, United StatesDepartment of Biological Sciences, Wright State University, 3640 Colonel Glenn Hwy, 235a BS, Dayton, OH 45435, United StatesDepartment of Biological Sciences, Wright State University, 3640 Colonel Glenn Hwy, 235a BS, Dayton, OH 45435, United States; Corresponding author.CRISPR-Cas9 gene editing has made it possible to specifically edit genes in a myriad of target cells. Here, a method for isoform-specific editing and clonal selection in Madin-Darby canine kidney (MDCK) epithelial cells is described in detail. This approach was used to address a long-standing question in virology of how adenovirus enters polarized epithelia from the apical surface. Our method relies on selecting two sgRNA sequences, cloning them into a suitable fluorescently labeled Cas9 vector system, and subsequently transfecting our MDCK epithelium and selecting isoform-specific Coxsackievirus and adenovirus receptor knockout clones. Utilization of this method is readily applicable to many other genetic targets in epithelial cells. • Simultaneous utilization of an sgRNA upstream and an sgRNA downstream of a target sequence allows for deletion of the intervening sequence, including whole exons. • Sorting of cells positive for fluorescent marker gene expression enhances the identification of partial and biallelic gene knockout. • PCR screening allows relatively fast and efficient determination of isoform-specific deletion.http://www.sciencedirect.com/science/article/pii/S2215016120303691Production of isoform-specific knockdown/knockout epithelialcell lines |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
James M. Readler Amal S. AlKahlout Abimbola O. Kolawole Katherine J.D.A. Excoffon |
spellingShingle |
James M. Readler Amal S. AlKahlout Abimbola O. Kolawole Katherine J.D.A. Excoffon Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9 MethodsX Production of isoform-specific knockdown/knockout epithelialcell lines |
author_facet |
James M. Readler Amal S. AlKahlout Abimbola O. Kolawole Katherine J.D.A. Excoffon |
author_sort |
James M. Readler |
title |
Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9 |
title_short |
Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9 |
title_full |
Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9 |
title_fullStr |
Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9 |
title_full_unstemmed |
Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9 |
title_sort |
production of isoform-specific knockdown/knockout madin–darby canine kidney epithelial cells using crispr/cas9 |
publisher |
Elsevier |
series |
MethodsX |
issn |
2215-0161 |
publishDate |
2020-01-01 |
description |
CRISPR-Cas9 gene editing has made it possible to specifically edit genes in a myriad of target cells. Here, a method for isoform-specific editing and clonal selection in Madin-Darby canine kidney (MDCK) epithelial cells is described in detail. This approach was used to address a long-standing question in virology of how adenovirus enters polarized epithelia from the apical surface. Our method relies on selecting two sgRNA sequences, cloning them into a suitable fluorescently labeled Cas9 vector system, and subsequently transfecting our MDCK epithelium and selecting isoform-specific Coxsackievirus and adenovirus receptor knockout clones. Utilization of this method is readily applicable to many other genetic targets in epithelial cells. • Simultaneous utilization of an sgRNA upstream and an sgRNA downstream of a target sequence allows for deletion of the intervening sequence, including whole exons. • Sorting of cells positive for fluorescent marker gene expression enhances the identification of partial and biallelic gene knockout. • PCR screening allows relatively fast and efficient determination of isoform-specific deletion. |
topic |
Production of isoform-specific knockdown/knockout epithelialcell lines |
url |
http://www.sciencedirect.com/science/article/pii/S2215016120303691 |
work_keys_str_mv |
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