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 library of cells comprising mutagenized mammalian genes, said mammalian genes comprising an integrated transcriptional terminator, at which site transcription terminates.  
     
     
         2 . The library of  claim 1 , wherein said cells are mouse cells.  
     
     
         3 . The library of  claim 1 , wherein said cells are mammalian stem cell.  
     
     
         4 . The library of  claim 3 , wherein said mammalian stem cells are embryonic stem cells.  
     
     
         5 . The library 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.  
     
     
         6 . The library of  claim 5 , wherein said vector further comprises a splice acceptor sequence.  
     
     
         7 . The library of  claim 5 , wherein said vector is a retroviral vector, said retroviral vector further comprising retroviral packaging sequences.  
     
     
         8 . The library of  claim 7 , wherein said retroviral vector comprises packaging and integration sequences from a Moloney murine leukemia virus sequence.  
     
     
         9 . The library of  claim 5 , wherein said vector further comprises a regulatory gene positioned for expression under the control of an endogenous promoter in a mammalian cell, wherein said promoter is operably linked to said regulatory gene upon integration of said vector into the genome of said mammalian cell.  
     
     
         10 . The library of  claim 9 , wherein said regulatory gene encodes a regulatory protein that modulates the expression of another gene.  
     
     
         11 . The library of  claim 10 , wherein said regulatory protein is a tetracycline repressor fused to VP 16 .  
     
     
         12 . The library of  claim 5 , wherein said vector further comprises a DNA sequence encoding a constitutively expressed marker gene, wherein said marker gene encodes a marker protein detectable in a mammalian cell.  
     
     
         13 . The library of  claim 5 , wherein said vector further comprises a recognition sequence recognized by a yeast VDE DNA endonuclease.  
     
     
         14 . The library of  claim 5 , wherein said vector further comprises a sequence which is recognized by a recombinase enzyme.  
     
     
         15 . A method for making a library of mammalian cells comprising mutagenized genes, said genes comprising an integrated transcriptional terminator, at which site transcription terminates, said method comprising introducing into said mammalian cells a transcription terminator, at which site transcription terminates, said introducing step being carried out under conditions which allow said transcription terminator to integrate into genes of said mammalian cells, thereby mutagenizing said genes.  
     
     
         16 . The method of  claim 15 , 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.  
     
     
         17 . The method of  claim 16 , wherein said vector further comprises a splice acceptor sequence.  
     
     
         18 . The method of  claim 16 , wherein said vector is a retroviral vector, said retroviral vector further comprising retroviral packaging sequences.  
     
     
         19 . The method of  claim 18 , wherein said retroviral vector comprises packaging and integration sequences from a Moloney murine leukemia virus sequence.  
     
     
         20 . The method of  claim 16 , 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.  
     
     
         21 . The method of  claim 20 , wherein said regulatory gene encodes a regulatory protein that modulates the expression of another gene.  
     
     
         22 . The method of  claim 21 , wherein said regulatory protein is a tetracycline repressor fused to a VP 16  transcription activation domain.  
     
     
         23 . The method of  claim 16 , 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.  
     
     
         24 . The method of  claim 16 , wherein said vector further comprises a sequence which is recognized by a recombinase enzyme.  
     
     
         25 . The method of  claim 16 , wherein said mammalian cells are mouse cells.  
     
     
         26 . The method of  claim 16 , wherein said mammalian cells are mammalian stem cells.  
     
     
         27 . The method of  claim 26 , wherein said mammalian stem cells are embryonic stem cells.  
     
     
         28 . A library of mutagenized mammalian genes, said mammalian genes comprising an integrated transcriptional terminator, at which site transcription terminates.