Many receptors, kinases and transcr

Many receptors, kinases and transcriptional factors regulate gene expression either directly or indirectly through signal transduction. By accessing these signal cascades, reporter gene technique offers great sensitivity for signal detection in cell-based assays. As we know, the sensitivity and efficiency of reporter gene systems are largely affected by both reporter gene and promoter upstream. In order to guarantee wide detecting windows and high resolutions, promoters with very low constitutive activities should be sought. This will ensure an optimal background and significant response in the presence of a stimulus. For reporter gene products, an appropriate level of stability is also vital. Long half-time may lead to an increased background resulting from excess accumulation of reporter gene encoded proteins. This could be caused by constitutive activation of promoters or lagging response to stimulations. Clearly, rational selection of both promoter and reporter gene according to the purpose and requirement of an assay is an indispensable step before HTS. Reporter genes that are commonly utilized today include green fluorescent protein (GFP), β-galactosidase, firefly luciferase, Renila luciferase and β-lactamase. The most widely used bioluminescent reporter genes are firefly luciferase from Photinuspyralis and Renilla luciferase derived from the sea pansyRenillareniformis. These bioluminescent reporter assays are of both high sensitivity and extraordinary accuracy. Because the readout is determined by efficiency of signal transduction and cellular physical/metabolic integrity, however, the false positive rate can be high18. Efforts in follow-up hit verification with secondary assays are generally required as a routine.