Gene reporter assay, kit and cells with improved sensitivity and/or specificity for determining the level of an extracellular signal

摘要

The present invention provides a reporter gene containing cell line with increased specificity and/or sensitivity for a particular extracellular signal of interest so that it can be used in a gene-reporter assay to accurately determine the presence and/or level of the extracellular signal of interest in the presence of other extracellular signals that are capable of activating the same signal transduction pathway as the extracellular signal of interest or that are capable of activating another signal transduction pathway capable of modulating the transcription of the reporter gene.

主权项

1. In a cell line stably transformed with a reporter gene construct comprising a nucleotide sequence encoding a reporter gene product operatively linked to a transcriptional control element that is activated as part of the NFκB signal transduction pathway activated by an interaction of a first extracellular signal with a first cell surface molecule or complex, wherein the transcriptional control element is one that bound by NFκB so as to activate said transcriptional control element and thereby induce or upregulate transcription of the reporter gene, the improvement whereby the sensitivity and/or the specificity of the response of the cell line to the extracellular signal is improved, wherein:
a) said transcriptional control element is a synthetic promoter consisting of a minimal promoter and a regulatory sequence, said minimal promoter being regulated by said regulatory sequence, said regulatory sequence consisting of a plurality of response elements specific for NFκB; and b) the cells of said cell line lack at least one functional second cell surface molecule or complex that, if present on the cell line, would interact with a second extracellular signal that is one of the principal signals known to activate the NFκB signal transduction pathway though said second cell surface molecule or complex, thereby modulating the transcription of the reporter gene and causing interference with the first extracellular signal when the cell line is used in a gene reporter assay.