| Autor: |
Nan Zuo, Lin Ma, Weitao Hu, Yongqiang Deng, Lanlan Wei, Qi Liu |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
Bio-Protocol, Vol 12, Iss 17 (2022) |
| Druh dokumentu: |
article |
| ISSN: |
2331-8325 |
| DOI: |
10.21769/BioProtoc.4506 |
| Popis: |
The main cellular pathways to repair DNA double-strand breaks (DSBs) and protect the integrity of the genome are homologous recombination (HR), non-homologous end-joining (NHEJ), and alternative end-joining (Alt-EJ). Polymerase theta-regulated Alt-EJ is an error-prone DSB repair pathway characterized by microhomology usage. Considering its importance in cancer treatment, technologies for detection of Alt-EJ in cancer cells may facilitate the study of the mechanisms of carcinogenesis and the development of new therapeutic targets. DSB reporter assay is the classical method for detecting Alt-EJ, which is primarily based on components of EJ2-puro cassette integration, I-SceI cleaving, and flow cytometry analysis. Here, we described an assay based on a modified I-Scel plasmid that can screen head and neck squamous cell carcinoma (HNSC) cells that were successfully transfected using selection medium with hygrovetine. We expect that this protocol will improve the fidelity and accuracy of reporter assays.Graphical abstract: Schematic overview of the workflow for establishment of Alt-EJ reporters. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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