Vectors and methods for the mutagenesis of mammalian genes

Abstract

Disclosed herein are methods for mutagenizing a mammalian gene, the methods involving introducing into a mammalian cell a retroviral vector which includes a splice acceptor sequence, a transcription termination sequence, and retroviral packaging and integration sequences, the introducing step being carried out under conditions which allow the vector to integrate into the genome of the cell. Also disclosed are retroviral vectors for use in these methods as well as methods for the use of mutagenized cells.

What is claimed is:  
     
         1 . A method for mutagenizing a gene, said method comprising introducing into a mammalian cell a transcription terminator, at which site transcription terminates, said introducing step being carried out under conditions which allow said transcription terminator to integrate into a gene of said mammalian cell, thereby mutagenizing said gene.  
     
     
         2 . The method of  claim 1 , wherein said transcription terminator is in a vector, said vector further comprising an integration sequence that mediates integration of said transcription terminator into said gene.  
     
     
         3 . The method of  claim 2 , wherein said vector further comprises a splice acceptor sequence.  
     
     
         4 . The method of  claim 2 , wherein said vector is a retroviral vector, said retroviral vector further comprising retroviral packaging sequences.  
     
     
         5 . The method of  claim 4 , wherein said retroviral vector comprises packaging and integration sequences from a Moloney murine leukemia virus sequence.  
     
     
         6 . The method of  claim 2 , wherein said vector further comprises a regulatory gene positioned for expression under the control of an endogenous promoter in said mammalian cell, said promoter being operably linked to said regulatory gene upon integration of said vector into the genome of said mammalian cell.  
     
     
         7 . The method of  claim 6 , wherein said regulatory gene encodes a regulatory protein that modulates the expression of another gene.  
     
     
         8 . The method of  claim 7 , wherein said regulatory protein is a tetracycline repressor fused to an activator protein.  
     
     
         9 . The method of  claim 8 , wherein said activator protein is a VP16 transcription activation domain.  
     
     
         10 . The method of  claim 2 , wherein said vector further comprises a DNA sequence encoding a constitutively expressed marker gene, said marker gene encoding a marker protein that is detectable in a mammalian cell.  
     
     
         11 . The method of  claim 10 , wherein said marker protein is a green fluorescent protein.  
     
     
         12 . The method of  claim 11 , wherein said green fluorescent protein has increased cellular fluorescence relative to the wild-type green fluorescent protein.  
     
     
         13 . The method of  claim 11 , wherein said green fluorescent protein is fused to a mammalian selectable marker protein.  
     
     
         14 . The method of  claim 13 , wherein said mammalian selectable marker protein is neomycin phosphotransferase.  
     
     
         15 . The method of  claim 2 , wherein said vector further comprises a recognition sequence recognized by a yeast VDE DNA endonuclease.  
     
     
         16 . The method of  claim 2 , wherein said vector further comprises a sequence which is recognized by a recombinase enzyme.  
     
     
         17 . The method of  claim 16 , wherein said sequence is a loxP sequence.  
     
     
         18 . The method of  claim 2 , wherein said mammalian cell is a mouse cell.  
     
     
         19 . The method of  claim 2 , wherein said mammalian cell is a mammalian stem cell.  
     
     
         20 . The method of  claim 19 , wherein said mammalian stem cell is an embryonic stem cell.