Microarrays of cells expressing defined cDNAs (original) (raw)

Nature volume 411, pages 107–110 (2001)Cite this article

Abstract

Genome and expressed sequence tag projects are rapidly cataloguing and cloning the genes of higher organisms, including humans. An emerging challenge is to rapidly uncover the functions of genes and to identify gene products with desired properties. We have developed a microarray-driven gene expression system for the functional analysis of many gene products in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of non-transfected cells. By printing sets of complementary DNAs cloned in expression vectors, we make microarrays whose features are clusters of live cells that express a defined cDNA at each location. Here we demonstrate two uses for our approach: as an alternative to protein microarrays for the identification of drug targets, and as an expression cloning system for the discovery of gene products that alter cellular physiology. By screening transfected cell microarrays expressing 192 different cDNAs, we identified proteins involved in tyrosine kinase signalling, apoptosis and cell adhesion, and with distinct subcellular distributions.

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References

  1. Schena, M., Shalon, D., Davis, R. W. & Brown, P. O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 (1995).
    Article ADS CAS Google Scholar
  2. Lockhard, D. J. et al. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nature Biotechnol. 14, 1675–1678 (1996).
    Article Google Scholar
  3. Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).
    Article ADS CAS Google Scholar
  4. Kino, T. et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J. Antibiot. (Tokyo) 40, 1256–1265 (1987).
    Article CAS Google Scholar
  5. Harding, M. W., Galat, A., Uehling, D. E. & Schreiber, S. L. A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 341, 758–760 (1989).
    Article ADS CAS Google Scholar
  6. Siekierka, J. J., Hung, S. H., Poe, M., Lin, C. S. & Sigal, N. H. A cytolsolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341, 755–757 (1989).
    Article ADS CAS Google Scholar
  7. Schreiber, S. L. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251, 283–287 (1991).
    Article ADS CAS Google Scholar
  8. Jacinto, E., Werlen, G. & Karin, M. Cooperation between Syk and Rac1 leads to synergistic JNK activation in T lymphocytes. Immunity 8, 31–41 (1998).
    Article CAS Google Scholar
  9. Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501 (1992).
    Article CAS Google Scholar
  10. Walczak, H. et al. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 16, 5386–5397 (1997).
    Article CAS Google Scholar
  11. Tandon, N. N., Kralisz, U. & Jamieson, G. A. Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion. J. Biol. Chem. 264, 7576–7583 (1989).
    Article CAS Google Scholar
  12. Simonsen, H. & Lodish, H. F. Cloning by function: expression cloning in mammalian cells. Trends Pharmacol. Sci. 15, 437–441 (1994).
    Article CAS Google Scholar
  13. Strausberg, R. L., Feingold, E. A., Klausner, R. D. & Collins, F. S. The mammalian gene collection. Science 286, 455–457 (1999).
    Article CAS Google Scholar
  14. Kawai, J. et al. Functional annotation of a full-length mouse cDNA collection. Nature 409, 685–690 (2001).
    Article ADS Google Scholar
  15. Yudate, H. T. et al. HUNT: launch of a full-length cDNA database from the Helix Research Institute. Nucleic Acids Res. 29,185–188 (2001).
    Article CAS Google Scholar
  16. Wiemann, S. et al. Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs. Genome Res. 11,422–435 (2001).
    Article CAS Google Scholar
  17. MacBeath, G. & Schreiber, S. L. Printing proteins as microarrays for high-throughput function determination. Science 289, 1760–1763 (2000).
    ADS CAS PubMed Google Scholar
  18. Lueking, A. et al. Protein microarrays for gene expression and antibody screening. Anal. Biochem. 270, 103–111 (1999).
    Article CAS Google Scholar
  19. de Wildt, R. M., Mundy, C. R., Gorick, B. D. & Tomlinson, I. M. Antibody arrays for high-throughput screening of antibody-antigen interactions. Nature Biotechnol. 9, 989–994 (2000).
    Article Google Scholar
  20. Morgenstern, J. P. & Land, H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990).
    Article CAS Google Scholar
  21. Sabatini, D. M. et al. Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling. Science 284, 1161–1164 (1999).
    Article ADS CAS Google Scholar

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Acknowledgements

We thank C. Wilson and T. Volkert for advice on use of the microarrayer, and K. McKernan and P. McEwan for the neurotransmitter receptor constructs. We also thank R. Young, N. Hacohen and members of the Sabatini laboratory for their support and suggestions; P. Kim and M. Gerlach for help with the manuscript; and Invitrogen for the full-length clones from the Genestorm collection. This research was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, the Whitehead Institute Fellows Program and Corning. Microscopy work was conducted at the W. M. Keck Foundation Biological Imaging facility at the Whitehead Institute.

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Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, 02142, Massachusetts, USA
    Junaid Ziauddin & David M. Sabatini

Authors

  1. Junaid Ziauddin
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  2. David M. Sabatini
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Corresponding author

Correspondence toDavid M. Sabatini.

Supplementary information

Clones from the Invitrogen Genestorm collection were used to create transfected cell microarrays expressing 192 cDNAs.

Genestorm cDNAs are in pcDNA3.1/GS vector.

**Table 2. Invitrogen Genestorm clones

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Ziauddin, J., Sabatini, D. Microarrays of cells expressing defined cDNAs.Nature 411, 107–110 (2001). https://doi.org/10.1038/35075114

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