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Establishment of Novel Reporter Cells Stably Maintaining Transcription Factor-driven Human Secreted Alkaline Phosphatase Expression

[ Vol. 19 , Issue. 3 ]

Author(s):

Riho Kurata, Asuka Kumagai, Xiaofeng Cui, Masamitsu Harada, Jun Nagai, Yasuhiro Yoshida, Kei-ichi Ozaki, Yoshimasa Tanaka and Tomo Yonezawa*   Pages 224 - 231 ( 8 )

Abstract:


Background: Transcriptional regulation is a very important and pivotal function in myriad biological responses. Thus, methods to determine transcriptional activity are required in not only basic medical research but also in drug discovery. We established novel reporter constructs using human secreted embryonic alkaline phosphatase (SEAP) and Epstein-Barr virus nuclear antigen (EBNA) 1, which can maintain constructs synchronized to host cell replication.

Methods: We established nuclear factor-kappa B (NFkB) or interferon regulatory factor (IRF) driven SEAP expression constructs and then, introduced them into culture cells.

Results: The cells maintain reporter constructs for a long period in the culture and produce SEAP into culture supernatant in response to each specific ligand such as lipopolysaccharide (LPS) and interferon- beta. Measuring SEAP with chemiluminescence makes it possible to get high standard dynamic range applying to high-throughput screening in drug discovery in both 96 and 384 well format. We can also use it to determine transcriptional activity in the cells transfected with expression plasmid or treated with various toll-like receptor (TLR) ligands in a concentration-dependent manner and time-dependent manner. Finally, we demonstrated drug screening using a number of natural products library.

Conclusion: We for the first time established the two novel reporter cells and validated their quality and accuracy enough to carry out drug screening.

Keywords:

EBNA1, SEAP, NFkB, IRF, drug screening, transcription activity.

Affiliation:

Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Center for Therapeutic Innovation, Gene Research Center for Fronties Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523-0022, School of Chemistry, Chemical Engineering and Life Sciences, School of Materials and Engineering, Wuhan University of Technology, 122 Loushi Rd, Wuhan, Hubei, Center for Therapeutic Innovation, Gene Research Center for Fronties Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523-0022, Center for Therapeutic Innovation, Gene Research Center for Fronties Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523-0022, Department of Immunology and Parasitology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807- 8555, Education and Research Center For Fundamental Pharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, Center for Therapeutic Innovation, Gene Research Center for Fronties Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523-0022, Center for Therapeutic Innovation, Gene Research Center for Fronties Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523-0022

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