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 transgenic non-human mammal, said mammal comprising a retroviral vector that comprises (i) a splice acceptor sequence, (ii) a transcription terminator, at which site transcription terminates, and (iii) retroviral packaging and integration sequences.  
     
     
         2 . The transgenic non-human mammal of    claim 1   , wherein said mammal is a mouse.  
     
     
         3 . The transgenic non-human mammal of    claim 1   , wherein said retroviral packaging and integration sequences are from a Moloney murine leukemia virus sequence.  
     
     
         4 . The transgenic non-human mammal of    claim 1   , wherein said retroviral vector further comprises a regulatory gene positioned for expression under the control of an endogenous promoter in the genome of said non-human mammal, whereby said promoter is operably linked to said regulatory gene upon integration of said vector into the genome of said mammal.  
     
     
         5 . The transgenic non-human mammal of    claim 4   , wherein said regulatory gene encodes a regulatory protein that modulates the expression of another gene.  
     
     
         6 . The transgenic non-human mammal of    claim 5   , wherein said regulatory protein is a tetracycline repressor fused to an activator protein.  
     
     
         7 . The transgenic non-human mammal of    claim 6   , wherein said activator protein is a VP16 transcription activation domain.  
     
     
         8 . The transgenic non-human mammal of    claim 1   , wherein said retroviral 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.  
     
     
         9 . The transgenic non-human mammal of    claim 8   , wherein said marker protein is a green fluorescent protein.  
     
     
         10 . The transgenic non-human mammal of    claim 9   , wherein said green fluorescent protein has increased cellular fluorescence relative to the wild-type green fluorescent protein.  
     
     
         11 . The trans genic non-human mammal of    claim 9   , wherein said green fluorescent protein is fused to a mammalian selectable marker protein.  
     
     
         12 . The transgenic non-human mammal of    claim 11   , wherein said mammalian selectable marker protein is neomycin phosphotransferase.  
     
     
         13 . The trans genic non-human mammal of    claim 1   , wherein said retroviral vector further comprises a recognition sequence recognized by a yeast VDE DNA endonuclease.  
     
     
         14 . The transgenic non-human mammal of    claim 1   , wherein said retroviral vector further comprises a sequence which is recognized by a recombinase enzyme.  
     
     
         15 . The transgenic non-human mammal of    claim 14   , wherein said sequence is a loxP sequence.