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

Summary: 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 c...

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Bibliographic Details
Published in:STAR Protocols
Main Authors: Natalie Rimmer, Ching-Yeu Liang, Ricardo Coelho, Monica Nunez Lopez, Francis Jacob
Format: Article
Language:English
Published: Elsevier 2023-06-01
Subjects:
Cell Culture
Flow Cytometry/Mass Cytometry
Cell Separation/Fractionation
Gene Expression
CRISPR
Online Access:http://www.sciencedirect.com/science/article/pii/S2666166723002721
Description
Summary:Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
ISSN:2666-1667

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