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A genome-wide functional assay of signal transduction in living mammalian cells

Nature Biotechnology volume 16, pages 1329–1333 (1998)Cite this article

Abstract

We describe a genome-wide functional assay for rapid isolation of cell clones and genetic elements responsive to specific stimuli. A promoterless β-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.

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Figure 1: Photomicrographs of CCF2 fluorescence from the unsorted cell library (A) before or (B) after 18 h of PMA/Tg stimulation.
Figure 2: Strategies for isolating PHA-stimulated (top row) and PHA-repressed (bottom row) T-cell gene-trap clones by flow cytometry.
Figure 3: Genomic Southern blot analysis of individual T-cell modulated clones.
Figure 4: Induction and repression graphs for six gene-trap clones that were modulated by T-cell activation.
Figure 5
Figure 6: Northern blot analysis.

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Acknowledgements

We thank Roger Tsien, Charles Zuker, Tom Curran, Kleanthis Xanthopoulos, and Timothy Rink for critical review of the manuscript. We also thank Bridgette Mandurrago for her technical assistance with DNA sequencing.

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  1. Aurora Biosciences Corporation, 11010 Torreyana Rd., San Diego, 92121, CA

    Mike Whitney, Edward Rockenstein, Greg Cantin, Tom Knapp, Gregor Zlokarnik, Pam Sanders, Kyle Durick, Frank F. Craig & Paul A. Negulescu

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Correspondence to Paul A. Negulescu.

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Whitney, M., Rockenstein, E., Cantin, G. et al. A genome-wide functional assay of signal transduction in living mammalian cells. Nat Biotechnol 16, 1329–1333 (1998). https://doi.org/10.1038/4302

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