Journal of Inflammation Research (Mar 2021)

Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs

  • Zhang S,
  • Luo T,
  • Wang J

Journal volume & issue
Vol. Volume 14
pp. 917 – 928

Abstract

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Shuyan Zhang, Tao Luo, Jinke Wang State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, People’s Republic of ChinaCorrespondence: Jinke WangState Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, People’s Republic of ChinaEmail [email protected]: NF-κB is a sequence-specific DNA-binding transcription factor that plays key roles in inflammation and cancer. It is well known that NF-κB is over-activated in these diseases. NF-κB inhibitors are therefore developed as promising drugs for these diseases. However, finding NF-κB inhibitors is dependent on effective screening platforms.Methods: For providing an easy and visualizable tool for screening NF-κB inhibitors, and other NF-κB-related studies, this study edited all five genes of NF-κB family (RELA, RELB, CREL, NF-κB1, NF-κB2) in three different cell lines (293T, HepG2, and PANC1) with both TALEN and CRISPR. The edited NF-κB genes were repaired by homology-dependent repair using a linear homologous donor containing ZsGreen coding sequence. The edit efficiency was thus directly evaluated by detecting cellular fluorescence. The editing efficiency was also confirmed by PCR detection of NF-κB-ZsGreen fused genes.Results: It was found that all genes were more efficiently edited by TALEN in all cells than CRISPR. The positive cells were then isolated from the TALEN-edited cell pool by flow cytometry. The purified positive cells were finally evaluated by regulating NF-κB activity with a known NF-κB inhibitor, BAY 11– 7082, and an NF-κB-targeting artificial microRNA, miR533. The results revealed that all the labeled NF-κB genes responded well to the two kinds of NF-κB activity regulators in all cell lines.Conclusion: This study thus obtained 15 cell lines with NF-κB-ZsGreen fused genes, which provide an easy and visualizable tool for screening NF-κB inhibitors and other NF-κB-related studies.Keywords: NF-κB, TALEN, edit, HDR, ZsGreen

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