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 promoterless beta-lactamase reporter gene was transfected into a human T-cell line to generate a living library of reporter-tagged clones. When loaded with a cell-permeable fluorogenic substrate, the cell library simultaneously reports the expression of a large number of endogenous genes. Flow cytometry was used to recover individual clones whose reporter-tagged genes were either induced or repressed following T-cell activation. Responsive clones were expanded and analyzed pharmacologically to identify patterns of regulation associated with specific genes. Although demonstrated using T cells, the genomic assay could be applied to map downstream transcriptional consequences for any propagating cell line in response to any stimulus of interest.
Whitney
M
M
Aurora Biosciences Corporation, San Diego, CA 92121, USA.
Rockenstein
E
E
Cantin
G
G
Knapp
T
T
Zlokarnik
G
G
Sanders
P
P
Durick
K
K
Craig
F F
FF
Negulescu
P A
PA
D002469:Cell Separation; D004789:Enzyme Activation; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D016678:Genome; D006801:Humans; D019169:Jurkat Cells; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D001618:beta-Lactamases / Q000235:genetics
D005456:Fluorescent Dyes; D019284:Thapsigargin; D001618:beta-Lactamases; D013755:Tetradecanoylphorbol Acetate