Novel retroviral vectors to facilitate expression screens in mammalian cells.
As tools for functional genomics, expression profiling and proteomics provide correlative data, while expression cloning screens can link genes directly to biological function. However, technical limitations of gene transfer, expression, and recovery of candidate genes have limited wider application of genome-wide expression screens. Here we describe the pEYK retroviral vectors, which maintain high titers and robust gene expression while addressing the major bottleneck of expression cloning--efficient candidate gene recovery. By exploiting schemes for enhanced PCR rescue or strategies for direct isolation of proviral DNA as plasmids in bacterial hosts, the pEYK vectors facilitate cDNA isolation from selected cells and enable rapid iteration of screens and genetic reversion analyses to validate gene candidates. These vectors have proven useful to identify genes linked to cell proliferation, senescence and apoptosis.
Koh
Eugene Y
EY
Whitehead Institute, Nine Cambridge Center, Cambridge, MA 02142, USA.
Chen
Tong
T
Daley
George Q
GQ
D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D015750:Genes, gag / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000254:growth & development; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics* / Q000254:growth & development; D014162:Transfection / Q000379:methods / Q000592:standards
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016044:Fusion Proteins, bcr-abl