Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs
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 wang...
Guardado en:
| Autores principales: | , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Dove Medical Press
2021
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/cdfdf55cbf0c4a7ab708212cb72c4c32 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| id |
oai:doaj.org-article:cdfdf55cbf0c4a7ab708212cb72c4c32 |
|---|---|
| record_format |
dspace |
| spelling |
oai:doaj.org-article:cdfdf55cbf0c4a7ab708212cb72c4c322021-12-02T18:23:04ZStable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs1178-7031https://doaj.org/article/cdfdf55cbf0c4a7ab708212cb72c4c322021-03-01T00:00:00Zhttps://www.dovepress.com/stable-cells-with-nf-kappab-zsgreen-fused-genes-created-by-talen-editi-peer-reviewed-article-JIRhttps://doaj.org/toc/1178-7031Shuyan 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 wangjinke@seu.edu.cnBackground: 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, ZsGreenZhang SLuo TWang JDove Medical Pressarticlenf-κbtalenedithdrzsgreenPathologyRB1-214Therapeutics. PharmacologyRM1-950ENJournal of Inflammation Research, Vol Volume 14, Pp 917-928 (2021) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
nf-κb talen edit hdr zsgreen Pathology RB1-214 Therapeutics. Pharmacology RM1-950 |
| spellingShingle |
nf-κb talen edit hdr zsgreen Pathology RB1-214 Therapeutics. Pharmacology RM1-950 Zhang S Luo T Wang J Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs |
| description |
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 wangjinke@seu.edu.cnBackground: 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 |
| format |
article |
| author |
Zhang S Luo T Wang J |
| author_facet |
Zhang S Luo T Wang J |
| author_sort |
Zhang S |
| title |
Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs |
| title_short |
Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs |
| title_full |
Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs |
| title_fullStr |
Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs |
| title_full_unstemmed |
Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs |
| title_sort |
stable cells with nf-κb-zsgreen fused genes created by talen editing and homology directed repair for screening anti-inflammation drugs |
| publisher |
Dove Medical Press |
| publishDate |
2021 |
| url |
https://doaj.org/article/cdfdf55cbf0c4a7ab708212cb72c4c32 |
| work_keys_str_mv |
AT zhangs stablecellswithnfkappabzsgreenfusedgenescreatedbytaleneditingandhomologydirectedrepairforscreeningantiinflammationdrugs AT luot stablecellswithnfkappabzsgreenfusedgenescreatedbytaleneditingandhomologydirectedrepairforscreeningantiinflammationdrugs AT wangj stablecellswithnfkappabzsgreenfusedgenescreatedbytaleneditingandhomologydirectedrepairforscreeningantiinflammationdrugs |
| _version_ |
1718378124451774464 |