Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining

We provide a protocol using non-homologous end joining to integrate an oligonucleotide sequence of a fluorescence protein at the CDH1 locus encoding for the epithelial glycoprotein E-cadherin. We describe steps for implementing the CRISPR-Cas9-mediated knock-in procedure by transfecting a cancer cel...

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Detalles Bibliográficos
Autores principales: Rimmer, Natalie, Liang, Ching-Yeu, Coelho, Ricardo, Lopez, Monica Nunez, Jacob, Francis
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10199306/
https://www.ncbi.nlm.nih.gov/pubmed/37178110
http://dx.doi.org/10.1016/j.xpro.2023.102305
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author Rimmer, Natalie
Liang, Ching-Yeu
Coelho, Ricardo
Lopez, Monica Nunez
Jacob, Francis
author_facet Rimmer, Natalie
Liang, Ching-Yeu
Coelho, Ricardo
Lopez, Monica Nunez
Jacob, Francis
author_sort Rimmer, Natalie
collection PubMed
description We provide a protocol using non-homologous end joining to integrate an oligonucleotide sequence of a fluorescence protein at the CDH1 locus encoding for the epithelial glycoprotein E-cadherin. We describe steps for implementing the CRISPR-Cas9-mediated knock-in procedure by transfecting a cancer cell line with a pool of plasmids. The EGFP-tagged cells are traced by fluorescence-activated cell sorting and validated on DNA and protein levels. The protocol is flexible and can be applied in principle to any protein expressed in a cell line. For complete details on the use and execution of this protocol, please refer to Cumin et al. (2022).(1)
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spelling pubmed-101993062023-05-21 Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining Rimmer, Natalie Liang, Ching-Yeu Coelho, Ricardo Lopez, Monica Nunez Jacob, Francis STAR Protoc Protocol We provide a protocol using non-homologous end joining to integrate an oligonucleotide sequence of a fluorescence protein at the CDH1 locus encoding for the epithelial glycoprotein E-cadherin. We describe steps for implementing the CRISPR-Cas9-mediated knock-in procedure by transfecting a cancer cell line with a pool of plasmids. The EGFP-tagged cells are traced by fluorescence-activated cell sorting and validated on DNA and protein levels. The protocol is flexible and can be applied in principle to any protein expressed in a cell line. For complete details on the use and execution of this protocol, please refer to Cumin et al. (2022).(1) Elsevier 2023-05-12 /pmc/articles/PMC10199306/ /pubmed/37178110 http://dx.doi.org/10.1016/j.xpro.2023.102305 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Rimmer, Natalie
Liang, Ching-Yeu
Coelho, Ricardo
Lopez, Monica Nunez
Jacob, Francis
Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining
title Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining
title_full Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining
title_fullStr Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining
title_full_unstemmed Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining
title_short Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining
title_sort generation of endogenously tagged e-cadherin cells using gene editing via non-homologous end joining
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10199306/
https://www.ncbi.nlm.nih.gov/pubmed/37178110
http://dx.doi.org/10.1016/j.xpro.2023.102305
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