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...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-06-01
|
| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723002721 |
| _version_ | 1797827794849234944 |
|---|---|
| author | Natalie Rimmer Ching-Yeu Liang Ricardo Coelho Monica Nunez Lopez Francis Jacob |
| author_facet | Natalie Rimmer Ching-Yeu Liang Ricardo Coelho Monica Nunez Lopez Francis Jacob |
| author_sort | Natalie Rimmer |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-09T12:54:05Z |
| format | Article |
| id | doaj.art-afdcb0e1a13442128cdfeef619702a44 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-09T12:54:05Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-afdcb0e1a13442128cdfeef619702a442023-05-14T04:29:50ZengElsevierSTAR Protocols2666-16672023-06-0142102305Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joiningNatalie Rimmer0Ching-Yeu Liang1Ricardo Coelho2Monica Nunez Lopez3Francis Jacob4Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, Switzerland; Corresponding authorOvarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, SwitzerlandOvarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, SwitzerlandOvarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, SwitzerlandOvarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel 4031, Switzerland; Hospital for Women, University Hospital Basel, Basel 4031, Switzerland; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2666166723002721Cell CultureFlow Cytometry/Mass CytometryCell Separation/FractionationGene ExpressionCRISPR |
| spellingShingle | Natalie Rimmer Ching-Yeu Liang Ricardo Coelho Monica Nunez Lopez Francis Jacob Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining STAR Protocols Cell Culture Flow Cytometry/Mass Cytometry Cell Separation/Fractionation Gene Expression CRISPR |
| 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 | Cell Culture Flow Cytometry/Mass Cytometry Cell Separation/Fractionation Gene Expression CRISPR |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723002721 |
| work_keys_str_mv | AT natalierimmer generationofendogenouslytaggedecadherincellsusinggeneeditingvianonhomologousendjoining AT chingyeuliang generationofendogenouslytaggedecadherincellsusinggeneeditingvianonhomologousendjoining AT ricardocoelho generationofendogenouslytaggedecadherincellsusinggeneeditingvianonhomologousendjoining AT monicanunezlopez generationofendogenouslytaggedecadherincellsusinggeneeditingvianonhomologousendjoining AT francisjacob generationofendogenouslytaggedecadherincellsusinggeneeditingvianonhomologousendjoining |