Methods and compositions for screening for altered cellular phenotypes

Abstract

The invention relates to methods and compositions useful for screening for altered cellular phenotypes using an inducible expression system to enrich for and detect the altered phenotypes and, more particularly, relates to screening libraries of candidate bioactive agents, for example, nucleic acids and peptides, in cells using an regulatable expression system to enrich for a subpopulation of cells having an altered phenotype due to the presence of a candidate bioactive agent.

1 - 56 . (canceled)  
     
     
         57 . A screening method comprising: 
 a) selecting from a parental population of mammalian cells a first population of cells that exhibit a phenotype in the presence of a test agent;    b) selecting from said first population of cells a second population of cells that do no exhibit said phenotype in the absence of a test agent; and    c) selecting from said second population of cells a third population of cells that exhibit said phenotype in the presence of said test agent.    
     
     
         58 . The screening method of  claim 57 , wherein said test agent is encoded by a first recombinant nucleic acid in said cell  
     
     
         59 . The screening method of clam  57 , wherein said test agent is a polypeptide.  
     
     
         60 . The screening method of  claim 57 , wherein said test agent is a cyclic polypeptide.  
     
     
         61 . The screening method of  claim 57 , wherein the presence of said test agent is modulated using an inducible system in which addition of an inducer increases production of said test agent.  
     
     
         62 . The screening method of  claim 57 , wherein the presence of said test agent is modulated using a repressible system in which addition of a repressor decreases production of said test agent.  
     
     
         63 . The screening method of  claim 57 , wherein said cells are cultured mammalian cells.  
     
     
         64 . The screening method of  claim 57 , wherein said selecting is done by fluorescence activated cell sorting (FACS).  
     
     
         65 . The screening method of  claim 57 , wherein said phenotype is detected by detecting a marker for said phenotype.  
     
     
         66 . The screening method of  claim 57 , wherein said phenotype is an optically detectable signal produced by a reporter protein.  
     
     
         67 . The screening method of  claim 66 , wherein said reporter protein is a fluorescent protein.  
     
     
         68 . The screening method of  claim 66 , wherein said reporter protein is encoded by a second recombinant nucleic acid in said cell.  
     
     
         69 . A screening method comprising: 
 a) selecting from a first population of cells a second population of cells, wherein said first population of cells exhibit a phenotype in the presence of a test agent and said second population of cells do no exhibit said phenotype in the absence of said test agent, and    b) selecting from said second population of cells a third population of cells that exhibit said phenotype in the presence of said test agent.    
     
     
         70 . The screening method of  claim 69 , wherein said test agent is encoded by a first recombinant nucleic acid in said cell  
     
     
         71 . The screening method of clam  69 , wherein said test agent is a polypeptide.  
     
     
         72 . The screening method of  claim 69 , wherein said test agent is a cyclic polypeptide.  
     
     
         73 . The screening method of  claim 69 , wherein the presence of said test agent is modulated using an inducible system in which addition of an inducer increases production of said test agent.  
     
     
         74 . The screening method of  claim 69 , wherein the presence of said test agent is modulated using a repressible system in which addition of a repressor decreases production of said test agent.  
     
     
         75 . The screening method of  claim 69 , wherein said cells are cultured mammalian cells.  
     
     
         76 . The screening method of  claim 69 , wherein said selecting is done by fluorescence activated cell sorting (FACS).  
     
     
         77 . The screening method of  claim 69 , wherein said phenotype is detected by detecting a marker for said phenotype.  
     
     
         78 . The screening method of  claim 69 , wherein said phenotype is an optically detectable signal produced by a reporter protein.  
     
     
         79 . The screening method of  claim 78 , wherein said reporter protein is a fluorescent protein.  
     
     
         80 . The screening method of  claim 78 , wherein said reporter protein is encoded by a second recombinant nucleic acid in said cell.