Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Heller, R.A., Schena, M., Chai, A., Shalon, D., Bedilion, T., Gilmore, J. et al. 1997. Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proc. Natl. Acad. Sci. USA 94: 2150– 2155.

    Article  Google Scholar 

  2. Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P.O., and Davis, R.W. et al. 1996. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. USA 93: 10614–10619.

    Article  Google Scholar 

  3. Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X.C., Stern, D. et al. 1996. Accessing genetic information with high-density arrays. Science 274: 610–614.

    Article  Google Scholar 

  4. Skarnes, W.C., Auerbach, B.A., and Joyner, A.L. 1992. A gene trap approach in mouse embryonic stem cells: the lacZ reporter is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice. Genes Dev. 6: 903–918.

    Article  Google Scholar 

  5. Forrester, L.M., Nagy, A., Sam, M., Watt, A., Stevenson, L., Berstein, A. et al. 1996. An induction gene trap screen in embryonic stem cells: identification of genes that respond to retenoic acid in vitro. Proc. Natl. Acad. Sci. USA 93: 1677–1682.

    Article  Google Scholar 

  6. Zlokarnik, G., Negulescu, P.A., Knapp, T.E., Mere, L., Burres, N., Feng., L. et al. 1998. Quantitation of transcription and clonal selection of single living cells using beta-lactamase as reporter. Science 279: 84–88.

    Article  Google Scholar 

  7. Wurst, W., Rossant, J., Prideaux, V., Kownacka, M., Joyner, A., Hill, D.P. et al. 1995. A large-scale gene-trap screen for insertional mutation in developmentally regulated genes in mice. Genetics 139: 889–899.

    Google Scholar 

  8. Gogos, J.A., Lowry, W., and Karayiorgou, M. 1997. Selection for retroviral insertions into regulated genes. J. Virol. 71: 1644–1650.

    PubMed Central  Google Scholar 

  9. Alberola-Ila, J., Takaki, S., Kerner, J.D., and Perlmutter, R.M. 1997. Differential signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 15: 125–154.

    Article  Google Scholar 

  10. Truneh, A., Albert, F., Goldstein, P., and Schmitt-Verhulst, A.M. 1985. Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature 313: 318–320.

    Article  Google Scholar 

  11. Thastrup, O., Cullen, P.J., Drobak, B.K., Hanley, M.R., and Dawson, A.P. 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc. Natl. Acad. Sci. USA 87: 2466– 2470.

    Article  Google Scholar 

  12. Takemura, H., Hughes, A.R., Thastrup, O., and Putney, J.W. 1989. Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. J. Biol. Chem. 264: 12266– 12271.

  13. Rosenstreich, D.L. and Mizel, S.B. 1979 . Signal requirements for T lymphocyte activation: replacement of macrophage function with phorbol myristic acetate. J. Immunol. 123: 1749–1755.

    Google Scholar 

  14. Clipstone, N.A. and Crabtree, G.R. 1992 . Identification of calcineurin as a key signaling enzyme in T-lymphocyte activation. Nature 357: 695– 697.

    Article  Google Scholar 

  15. Elliot, J.F., Lin, Y., Mizel, S.B., Bleackley, R.C., Harnish, D.G., and Paetkau, V. 1984. Induction of interleukin 2 messenger RNA inhibited by cyclosporin A. Science 226: 1439– 1443.

    Article  Google Scholar 

  16. Negulescu, P.A., Shastri, N., and Calahan, M.D. 1994.Intracellular calcium dependence of gene expression in single T lymphocytes. Proc. Natl. Acad. Sci. USA 91: 2873–2877.

    Article  Google Scholar 

  17. Fiering, S. and Al., E. 1990. Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 4: 1823–1830.

    Article  Google Scholar 

  18. Frohman, M.A., Dush, M.K., and Martin, G.R. 1988. Rapid production of full length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998 –9002.

    Article  Google Scholar 

  19. Nagasawa, M., Melamed, I., Kupfer, A., Gelfand, E.W., and Lucas, J.J. 1997. Rapid nuclear translocation and increased activity of cyclin-dependent kinase 6 after T-cell activation. J. Immunol. 158: 5146–5154 .

    Google Scholar 

  20. Mages, H.W., Stamminger, T., Rilke, O., Bravo, R., and Kroczek, R.A. 1993. Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T-cells. Int. Immunol. 5: 63–70.

    Article  Google Scholar 

  21. Patwardhan, S., Gashler, A., Siegel, M.G., Chang, L.C., Joseph, L.J., Shows, T.B. et al. 1991. EGR3, a novel member of the Egr family of genes encoding immediate-early transcription factors. Oncogene 6: 917–928.

    Google Scholar 

  22. Korchak, H.M., Kane, L.H., and Rossi, M.W. 1994. Long chain acyl coenzyme A and signaling in neutrophils. An inhibitor of acyl coenzyme A synthetase, triacsin C, inhibits superoxide anion generation and degranulation of human neutrophils. J. Biol. Chem. 269: 30281– 30287.

    Google Scholar 

  23. Faergeman, M.J. and Knudsen, J. 1997 . Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling. Biochem. J. 323: 1–12.

    Article  Google Scholar 

  24. Townley, D.J., Avery, B.J., Rosen, B., and Skarnes, W.C. 1997 .Rapid sequence analysis of gene trap integrations to generate a resourse of insertional mutations in mice. Genome Res. 7: 293–298.

    Article  Google Scholar 

  25. Reddy, S., Rayburn, H., von Melcher, H., and Ruley, H.E. 1992. Fluorescence-activated sorting of titopotent embryonic stem cells expressing developmentally regulated lacZ fusion genes. Proc. Natl. Acad. Sci. USA 89: 6721– 6725.

    Article  Google Scholar 

  26. Nolan, G.P., Fiering, S., Nicolas, J.F., and Herzenberg, L.A. 1988. Flourescence-activated cell analysis and sorting of viable mammalian cells based on β-D-galactosidase activity after transduction of E. coli lacZ. Proc. Natl. Acad. Sci. USA 85: 2603–2607.

    Article  Google Scholar 

  27. Chowdury, H., Bonaldo, P., Torres, M., Stoykova, A., and Gruss, P. 1997. Evidence for the satochastic integration of gene trap vectors into the mouse germline. Nucleic Acids Res. q 25: 1531–1536.

    Article  Google Scholar 

  28. Miller, S.A., Dykes, D.D., and Polesky, H.F. 1998. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16: 1215.

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  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

Authors
  1. Mike Whitney
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Edward Rockenstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Greg Cantin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tom Knapp
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gregor Zlokarnik
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pam Sanders
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kyle Durick
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Frank F. Craig
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Paul A. Negulescu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul A. Negulescu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/4302

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing