A high-throughput alphavirus-based expression cloning system for mammalian cells.
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.
Koller
D
D
Cytos Biotechnology AG, Wagistr. 21, CH-8952 Schlieren-Zürich, Switzerland.
Ruedl
C
C
Loetscher
M
M
Vlach
J
J
Oehen
S
S
Oertle
K
K
Schirinzi
M
M
Deneuve
E
E
Moser
R
R
Kopf
M
M
Bailey
J E
JE
Renner
W
W
Bachmann
M F
MF
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
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D008024:Ligands; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins