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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.

Original publication

DOI

10.1038/nbt0901-851

Type

Journal article

Journal

Nat Biotechnol

Publication Date

09/2001

Volume

19

Pages

851 - 855

Keywords

Animals, Antibodies, Monoclonal, Cell Line, Cell Membrane, Cell Separation, Cells, Cultured, Cloning, Molecular, Cricetinae, DNA, Complementary, Flow Cytometry, Green Fluorescent Proteins, Ligands, Luminescent Proteins, Mice, Models, Biological, Protein Binding, Reverse Transcriptase Polymerase Chain Reaction, Sindbis Virus, Time Factors