A high-throughput alphavirus-based expression cloning system for mammaliancells

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identificat ion of genes encoding a functional activity such as binding of a defined li gand. 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 so rting (FACS). Replication-competent, infective SIN replicon particles harbo ring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal anti bodies or Fc-fusion molecules could be isolated and sequenced. Moreover, us ing the same viral libraries, a plaque-lift assay was established that allo wed the identification of secreted, intracellular, and membrane proteins.