title: A high-throughput alphavirus-based expression cloning system for mammalian cells.
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.
authors.affiliation: Cytos Biotechnology AG, Wagistr. 21, CH-8952 Schlieren-Zürich, Switzerland.
authors.forename: D
authors.initials: D
authors.lastname: Koller
authors.forename: C
authors.initials: C
authors.lastname: Ruedl
authors.forename: M
authors.initials: M
authors.lastname: Loetscher
authors.forename: J
authors.initials: J
authors.lastname: Vlach
authors.forename: S
authors.initials: S
authors.lastname: Oehen
authors.forename: K
authors.initials: K
authors.lastname: Oertle
authors.forename: M
authors.initials: M
authors.lastname: Schirinzi
authors.forename: E
authors.initials: E
authors.lastname: Deneuve
authors.forename: R
authors.initials: R
authors.lastname: Moser
authors.forename: M
authors.initials: M
authors.lastname: Kopf
authors.forename: J E
authors.initials: JE
authors.lastname: Bailey
authors.forename: W
authors.initials: W
authors.lastname: Renner
authors.forename: M F
authors.initials: MF
authors.lastname: Bachmann
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012845:Sindbis Virus / Q000235:genetics*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D008024:Ligands; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins