Novel Reporter Systems Monitoring NFkB activation Can Be Applied toHigh-Throughput Screening.

Abstract

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 a novel reporter construct using human secreted embryonic alkaline phosphatase (SEAP) and Epstein-Barr virus nuclear antigen (EBNA) 1, which can maintain constructs synchronized to host cell replication. We established nuclear factor-kappa B (NFkB) driven SEAP expression construct and then, introduced it into culture cells. The cells maintain the reporter construct for a long period in the culture and produce SEAP into culture supernatant in response to a specific ligand, lipopolysaccharide. Measuring SEAP with chemiluminescence makes it possible to get high standard dynamic range applying to high-throughput screening in drug discovery.