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.
Subject(s)
Cloning, Molecular/methods , Sindbis Virus/genetics , Animals , Antibodies, Monoclonal/metabolism , Cell Line , Cell Membrane/metabolism , Cell Separation , Cells, Cultured , Cricetinae , DNA, Complementary/metabolism , Flow Cytometry , Green Fluorescent Proteins , Ligands , Luminescent Proteins/metabolism , Mice , Models, Biological , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
Hitherto, there have been few means to assess the immediate and secondary sequellae following the anatomic upheaval of buccopharyngeal carcinological exeresis. The functional recovery test, the various criteria of which are analyzed, to which 420 patients were subjected, makes it possible to record, for the 123 complete files, the regular evolution of the progress of each patient over a period of 12 months and beyond, which is stimulating and leads us to propose a more systematic handling of such operated patients by the team of phoniatricians and speech therapists.