Bi-directionally cloned random cDNA expression vector libraries, compositions and methods of use

Abstract

The present invention provides random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in sense and antisense orientation, which are useful for the delivery and expression of a combination of genetic effector types to host cells. Methods for producing these libraries through bi-directional cloning of random cDNAs are also provided. Also provided herein are methods of using these libraries to screen for agents capable of modulating cell phenotype in desirable ways.

1 - 20 . (canceled)  
     
     
         21 . A bi-directionally cloned cDNA expression vector library comprising: 
 a) a first plurality of expression vectors, each comprising: 
 i. a promoter that is active in a mammalian cell; and  
 ii. a cDNA fragment that is operably linked to said promoter in a sense orientation; and  
   b) a second plurality of expression vectors, each comprising: 
 i. said promoter; and  
 ii. a cDNA fragment that is operably linked to said promoter in an antisense orientation.  
   
     
     
         22 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein said expression vectors are retroviral expression vectors.  
     
     
         23 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein said expression vectors comprises cDNA fragments from a mammalian source.  
     
     
         24 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein said expression vectors further comprise a coding sequence for a reporter protein, operably linked to said promoter.  
     
     
         25 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein, wherein said reporter protein is a green fluorescent protein (GFP).  
     
     
         26 . The bi-directionally cloned cDNA expression vector library of  claim 24 , wherein said coding sequence and said cDNA fragment are separated by an internal ribosomal entry site (IRES).  
     
     
         27 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein said promoter is a constitutive promoter.  
     
     
         28 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein said promoter is an inducible promoter.  
     
     
         29 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein said library comprises from 10 3  to 10 9  vectors.  
     
     
         30 . The bi-directionally cloned cDNA expression vector library of  claim 21 , wherein said library is made by a method comprising: 
 contacting mRNA molecules with a primer under conditions to produce a double-stranded cDNA, said primer having a site for a first restriction endonuclease;    cleaving said double stranded cDNA to produce cDNA fragments;    ligating a double-stranded adaptor to each end of said cDNA fragments to produce adaptor-modified cDNAs, said double stranded adaptor comprising a site for a second restriction endonuclease;    contacting said adaptor-modified cDNAs with said first restriction endonuclease and said second restriction endonuclease to produce digested cDNAs; and, 
 ligating said digested cDNAs into vectors to produce a library of vectors;  
 to make said library.  
   
     
     
         31 . A population of cells comprising the bi-directionally cloned cDNA expression vector library of  claim 21 .  
     
     
         32 . The population of cells of  claim 21 , wherein said cells are mammalian cells.