Compositions and methods for non-targeted activation of endogenous genes

Abstract

The present invention is directed generally to activating gene expression or causing over-expression of a gene by recombination methods in situ. The invention also is directed generally to methods for expressing an endogenous gene in a cell at levels higher than those normally found in the cell. In one embodiment of the invention, expression of an endogenous gene is activated or increased following integration into the cell, by non-homologous or illegitimate recombination, of a regulatory sequence that activates expression of the gene. The invention also provides methods for the identification, activation, isolation, and/or expression of genes undiscoverable by current methods since no target sequence is necessary for integration. Thus, by the present invention, endogenous genes, including those associated with human disease and development, may be activated and isolated without prior knowledge of the sequence, structure, function, or expression profile of the genes.

1 - 57 . (canceled)  
     
     
         58 . A method for over-expressing a desired protein encoded by a desired endogenous gene or portion thereof in a cell introduced into a non-human animal, said method comprising: 
 (a) introducing a vector comprising a transcriptional regulatory sequence into one or more cells in vitro;    (b) maintaining the one or more cells containing the introduced vector from step (a) under conditions appropriate for non-homologous recombination of the vector with the genome of the one or more cells thereby producing one or more non-homologously recombinant cells, wherein the transcriptional regulatory sequence on the vector is operably linked to an endogenous gene thereby over-expressing the endogenous gene and protein encoded by said endogenous gene;    (c) screening said one or more cells from step (b) for expression of a desired endogenous gene;    (d) isolating and cloning said cell from step (c) expressing said desired endogenous gene; and    (e) introducing said non-homologously recombinant cell of step (d) into a non-human animal and maintaining the non-homologously recombinant cell in said animal under conditions appropriate for over-expression of said desired protein encoded by said desired endogenous cellular gene or portion thereof.    
     
     
         59 . The method of  claim 58 , wherein said transcriptional regulatory sequence is a promoter.  
     
     
         60 . The method of  claim 59 , wherein said promoter is a viral promoter.  
     
     
         61 . The method of  claim 60 , wherein said viral promoter is the cytomegalovirus immediate early promoter.  
     
     
         62 . The method of  claim 59 , wherein said promoter is a non-viral promoter.  
     
     
         63 . The method of  claim 59 , wherein said promoter is inducible.  
     
     
         64 . The method of  claim 58 , further comprising introducing double strand breaks into the genomic DNA of said cell prior to or simultaneously with integration of said vector.  
     
     
         65 . The method of  claim 58 , wherein said vector is linear.  
     
     
         66 . The method of  claim 58 , wherein said endogenous cellular gene encodes a transmembrane protein.  
     
     
         67 . The method of  claim 58 , wherein said promoter is operably linked to a splice donor sequence on the vector and wherein the splice donor is spliced to a splice acceptor in the endogenous gene that encodes the desired protein.  
     
     
         68 . The method of  claim 58 , wherein said animal is a mammal.  
     
     
         69 . The method of  claim 58 , wherein said mammal is a human.