Methods for identifying nucleic acid sequences encoding agents that affect cellular phenotypes

Abstract

Methods for identifying nucleic acid sequences that affect a cellular phenotype are disclosed. The method uses a reporter gene whose level of expression correlates with the phenotype in conjunction with a method or device for measuring the level of reporter expression. An expression library is introduced into the cells, and those cells exhibiting changes in reporter expression level are selected. Expression library inserts from the selected cells are isolated, thereby providing a sub-library enriched for sequences that affect the phenotype reflected by the reporter. Further rounds of sub-library introduction and cell selection may be carried out to provide additional enrichment. Sequences identified using this method may be used to ascertain the identity of additional molecules involved in generating the cellular phenotype.

What is claimed is:  
     
         1 . An assay for a nucleic acid that exerts an effect on a cellular pathway, comprising the steps of: 
 (a) providing an initial cell population transformed with a reporter construct under the control of a cis-regulatory element related to said cellular pathway;    (b) transfecting said cell population with an expression library;    (c) evaluating reporter expression levels in said cells transfected with said expression library;    (d) selecting a subpopulation of cells with a desired reporter expression level; and    (e) obtaining a sublibrary from said subpopulation of cells, wherein said sublibrary contains at least one nucleic acid encoding a perturbagen that exerts an effect on said cellular pathway.    
     
     
         2 . The method of  claim 1 , wherein said initial cell population is a mammalian cell population.  
     
     
         3 . The method of  claim 1 , wherein said step of evaluating comprises fluorescence activated cell sorter analysis of a fluorescent reporter.  
     
     
         4 . The method of  claim 1 , wherein said effect is activation of said cellular pathway.  
     
     
         5 . The method of  claim 1 , wherein said effect is inactivation of said cellular pathway.  
     
     
         6 . The method of  claim 1 , wherein said cellular pathway is growth-related.  
     
     
         7 . The method of  claim 1 , wherein said perturbagen is an RNA perturbagen.  
     
     
         8 . The method of  claim 7 , wherein said RNA perturbagen is a non-antisense RNA perturbagen.  
     
     
         9 . The method of  claim 7 , wherein said RNA perturbagen is a randomly generated RNA perturbagen.  
     
     
         10 . The method of  claim 9 , wherein said RNA perturbagen is an antisense perturbagen.