Vectors for gene mutagenesis and gene discovery

Abstract

Novel vectors are described that incorporate, inter alia, a novel 3′ gene trap cassette which can be used to efficiently trap and identify previously unknown cellular genes. Vectors incorporating the described 3′ gene trap cassette find particular application in gene discovery and in the production of mutated cells and animals.

1 - 21 . (canceled)  
     
     
         22 . A vector comprising: 
 a) a 5′ gene trap cassette, comprising in operable combination: 
 1) a splice acceptor;  
 2) a first exon located 3′ to said splice acceptor, said first exon encoding a marker enabling the identification of a cell expressing said exon; and  
 3) a polyadenylation sequence defining the 3′ end of said first exon;  
   b) a sequence encoding a self-cleaving RNA located 3′ to said polyadenylation sequence; and    c) a 3′ gene trap cassette located 3′ to said sequence encoding a self-cleaving RNA and comprising in operable combination: 
 1) a first promoter;  
 2) a second exon located 3′ from and expressed by said promoter, said second exon not encoding an activity conferring antibiotic resistance;  
 3) a splice donor sequence defining the 3′ region of the second exon; and  
 wherein said vector does not encode a promoter mediating the expression of said first exon, and wherein said vector does not encode a sequence that mediates the polyadenylation of an mRNA transcript encoded by said second exon and expressed by said first promoter.  
   
     
     
         23 . The vector according to  claim 22  wherein said first exon additionally encodes an internal ribosome entry site operatively positioned between said splice acceptor and an initiation codon of said first exon.  
     
     
         24 . The vector of  claim 22  wherein said second exon and said splice donor sequence are derived from a naturally occurring eukaryotic gene.  
     
     
         25 . The vector of  claim 22  further comprising a mutagenic mini-exon sequence operatively positioned upstream from said splice acceptor site.  
     
     
         26 . The vector of  claim 22  further comprising in the region between said polyadenylation sequence and said first promoter at least one mutagenesis enhancer selected from a transcription termination sequence, a 3′ terminal exon, and an exon that changes the reading frame.  
     
     
         27 . The vector of  claim 22  wherein said first exon encodes at least one marker selected from a marker conferring antibiotic resistance, a marker conferring antibiotic sensitivity, an enzymatic marker, a recombinase, and a fluorescent marker.  
     
     
         28 . The vector of  claim 27  wherein the at least one marker confers neomycin resistance.  
     
     
         29 . A vector comprising: 
 a) a first cassette, comprising in operable combination: 
 1) a first promoter; and  
 2) a marker gene expressed by said first promoter;  
   b) a sequence encoding a self-cleaving RNA located 3′ to said marker gene; and    c) a 3′ gene trap cassette, comprising in operable combination: 
 1) a second promoter;  
 2) an exon located 3′ from and expressed by said second promoter, said exon not encoding an activity conferring antibiotic resistance;  
 3) a splice donor sequence defining the 3′ region of the exon; and  
 wherein said vector does not encode a sequence that mediates the polyadenylation of an mRNA transcript encoded by said exon.  
   
     
     
         30 . A vector comprising: 
 a) a 5′ gene trap cassette comprising in operable combination: 
 1) a splice acceptor;  
 2) a first exon located 3′ to said splice acceptor, said first exon encoding a marker enabling the identification of a cell expressing said exon; and  
 3) a polyadenylation sequence defining the 3′ end of said first exon;  
   b) a sequence encoding a self-cleaving RNA located 3′ to said polyadenylation sequence; and    c) a 3′ gene trap cassette located 3′ to said sequence encoding a self-cleaving RNA and comprising in operable combination: 
 1) a first promoter;  
 2) a second exon located 3′ from and expressed by said promoter, said second exon being of non-prokaryotic origin;  
 3) a splice donor sequence defining the 3′ region of the exon; and  
 wherein said vector does not encode a promoter mediating the expression of said first exon, and wherein said vector does not encode a sequence that mediates the polyadenylation of an mRNA transcript encoded by said second exon and expressed by said first promoter.  
   
     
     
         31 . An infectious retrovirus comprising a vector according to any one of claims  22 ,  29 , or  30 .  
     
     
         32 . A method of trapping a gene in a eukaryotic target cell comprising introducing a retrovirus according to  claim 31  into said target cell in vitro.  
     
     
         33 . A method of trapping a gene in a eukaryotic target cell comprising introducing a vector according to any one of claims  22 ,  29 , or  30  into said cell in vitro, wherein said vector is introduced into said target cell by at least one method selected from electroporation, viral infection, retrotransposition, microinjection, and transfection.  
     
     
         34 . A eukaryotic cell which has a vector according to any one of claims  22 ,  29 , or  30  incorporated into its genome, wherein the vector was introduced into the eukaryotic cell or an ancestor of the eukaryotic cell in vitro.  
     
     
         35 . A non-human animal comprising the eukaryotic cell of  claim 34 .  
     
     
         36 . A method of activating the expression of a naturally occurring gene in a cell comprising introducing a vector according to any one of claims  22 ,  29 , or  30  into said cell in vitro.  
     
     
         37 . The method of  claim 36  wherein said cell is a mammalian cell.  
     
     
         38 . The method of  claim 37  wherein said mammalian cell is selected from a human cell and a mouse cell.  
     
     
         39 . A method of altering the expression of a cellularly encoded gene in a eukaryotic cell comprising introducing a vector into said cell, wherein said vector comprises in operable combination: 
 a) a sequence encoding a self-cleaving RNA; and    b) a 3′ gene trap cassette located 3′ to said sequence encoding a self-cleaving RNA and comprising in operable combination: 
 1) a promoter;  
 2) a first exon located 3′ from and expressed by said promoter, said first exon not encoding an activity conferring antibiotic resistance; and  
 3) a splice donor sequence defining the 3′ region of the first exon;  
 wherein said vector is non-homologously incorporated into the genome of the eukaryotic target cell and wherein said splice donor sequence of the transcript encoded by said first exon is spliced to a splice acceptor sequence of an exon of said cellularly encoded gene.  
   
     
     
         40 . The method of  claim 39  wherein said non-homologously incorporated vector is part of a retroviral vector that has nonspecifically integrated into the genome of the eukaryotic target cell.  
     
     
         41 . The method of  claim 40  wherein said first exon is selected from an exon not encoded by the target cell genome and an exon not normally expressed by the target cell genome.  
     
     
         42 . A method of obtaining novel eukaryotic polynucleotide sequence information comprising 
 a) introducing into a eukaryotic cell a vector comprising in operable combination: 
 1) a sequence encoding a self-cleaving RNA; and  
 2) a 3′ gene trap cassette located 3′ to said sequence encoding a self-cleaving RNA and comprising in operable combination: 
 i) a promoter;  
 ii) a first exon located 3′ from and expressed by said promoter, said first exon not encoding an activity conferring antibiotic resistance;  
 iii) a splice donor sequence defining the 3′ region of the first exon;  
 
   b) maintaining the cell under conditions allowing the nontargeted integration of the vector into the genome of the cell;    c) obtaining the chimeric transcript resulting from the splicing of said first exon from said 3′ gene trap cassette to a second exon encoded by the genome of said eucaryotic cell; and    d) reverse transcribing said chimeric transcript in vitro to produce a cDNA template; and    e) determining the polynucleotide sequence of the cDNA from step d.