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A high-throughput alphavirus-based expression cloning system for mammalian cells

Nature Biotechnology volume 19, pages 851–855 (2001)Cite this article

Abstract

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

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Figure 1: Scheme of the SIN replicon vectors and the expression screening strategy.
Figure 2: Highly efficient and controlled infection with single SIN replicon particles.
Figure 3: Sorting and recovery efficiency of SIN replicon particle–infected cells.
Figure 4: Single-step isolation of ligands for monoclonal antibodies or Fc-fusion molecules.
Figure 5: Isolation of genes encoding secreted, intracellular, and membrane proteins.

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Acknowledgements

We thank Dr Gary Jennings and Mark Dyer for carefully reading the manuscript and Prof. Sondra Schlesinger for many helpful discussions and for providing the plasmids pSinRep5 and pDH-EB. This article is dedicated to the memory of Prof. James E. Bailey, who passed away during the preparation of the manuscript.

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Authors and Affiliations

  1. Cytos Biotechnology AG, Wagistr. 21, Schlieren-Zürich, CH-8952, Switzerland

    Daniel Koller, Christiane Ruedl, Marcel Loetscher, Jaromir Vlach, Stefan Oehen, Karin Oertle, Michel Schirinzi, Estelle Deneuve, Wolfgang Renner & Martin F. Bachmann

  2. Institute of Biotechnology, Zurich, ETH, Switzerland

    Daniel Koller & James E. Bailey

  3. Basel Institute for Immunology, Basel, Switzerland

    Manfred Kopf

  4. IBR-Inc., Waengi, CH-9545, Switzerland

    Rene Moser

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  1. Daniel Koller
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Correspondence to Martin F. Bachmann.

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Koller, D., Ruedl, C., Loetscher, M. et al. A high-throughput alphavirus-based expression cloning system for mammalian cells. Nat Biotechnol 19, 851–855 (2001). https://doi.org/10.1038/nbt0901-851

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