A GENOME-WIDE FUNCTIONAL ASSAY OF SIGNAL-TRANSDUCTION IN LIVING MAMMALIAN-CELLS

We describe a genome-wide functional assay for rapid isolation of cell clones and genetic elements responsive to specific stimuli. A promote rless p-lactamase reporter gene was transfected into a human T-cell li ne to generate a living library of reporter-tagged clones. When loaded with a cell-permeable fluorogenic substrate, the cell library simulta neously reports the expression of a large number of endogenous genes. Flow cytometry was used to recover individual clones whose reporter-ta gged genes were either induced or repressed following T-ceIl activatio n. Responsive clones were expanded and analyzed pharmacologically to i dentify patterns of regulation associated with specific genes. Althoug h demonstrated using T cells, the genomic assay could be applied to ma p downstream transcriptional consequences for any propagating cell lin e in response to any stimulus of interest.