Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same

Abstract

Positive-negative selector (PNS) vectors are provided for modifying a target DNA sequence contained in the genome of a target cell capable of homologous recombination. The vector comprises a first DNA sequence which contains at least one sequence portion which is substantially homologous to a portion of a first region of a target DNA sequence. The vector also includes a second DNA sequence containing at least one sequence portion which is substantially homologous to another portion of a second region of a target DNA sequence. A third DNA sequence is positioned between the first and second DNA sequences and encodes a positive selection marker which when expressed is functional in the target cell in which the vector is used. A fourth DNA sequence encoding a negative selection marker, also functional in the target cell, is positioned 5′ to the first or 3′ to the second DNA sequence and is substantially incapable of homologous recombination with the target DNA sequence. The invention also includes transformed cells containing at least one predetermined modification of a target DNA sequence contained in the genome of the cell. In addition, the invention includes organisms such as non-human transgenic animals and plants which contain cells having predetermined modifications of a target DNA sequence in the genome of the organism.

1 - 90 . (canceled)  
     
     
         91 . An isolated mouse embryonic stem cell having a genome comprising a positive selection marker DNA sequence inserted into a non-selectable gene by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.  
     
     
         92 . The isolated embryonic stem cell of  claim 91  wherein said non-selectable gene is a gene of the immune system.  
     
     
         93 . The isolated embryonic stem cell of  claim 92 , wherein said genome further comprises a heterologous protein coding sequence other than a positive selection marker DNA sequence.  
     
     
         94 . The isolated embryonic stem cell of  claim 93 , wherein said heterologous protein coding sequence is a human gene.  
     
     
         95 . The isolated embryonic stem cell of  claim 94 , wherein said human gene is a gene of the human immune system.  
     
     
         96 . The isolated embryonic stem cell of  claim 95 , wherein said genome is without a positive screening marker DNA sequence.  
     
     
         97 . The isolated embryonic stem cell of  claim 96 , wherein said genome is without a bacterial tRNA suppressor gene.  
     
     
         98 . An isolated mouse embryonic stem cell having a genome comprising a human gene in place of a corresponding mouse gene, wherein said human gene is inserted into said genome by a homologous recombination event between said genome and a targeting vector, without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.  
     
     
         99 . The isolated mouse embryonic stem cell of  claim 98 , wherein said human gene is a gene of the immune system.  
     
     
         100 . The isolated embryonic stem cell of  claim 91  produced by the steps of: 
 (a) transforming a population of cells with a positive-negative selection (PNS) vector; and    (b) identifying said cell by selecting for cells containing said positive selection marker and against cells containing said negative selection marker;    wherein said PNS vector comprises: 
 (1) a first homologous vector DNA sequence capable of homologous recombination with a first region of said target DNA sequence;  
 (2) a positive selection marker DNA sequence capable of conferring a positive selection characteristic in said cells;  
 (3) a second homologous vector DNA sequence capable of homologous recombination with a second region of said target DNA sequence; and  
 (4) a negative selection marker DNA sequence, capable of conferring a negative selection characteristic in said cells, but substantially incapable of homologous recombination with said target DNA sequence;  
   wherein the spatial order of said sequences in said PNS vector is: said first homologous vector DNA sequence, said positive selection marker DNA sequence, said second homologous vector DNA sequence and said negative selection marker DNA sequence as shown in  FIG. 1 ;    wherein the 5′-3′ orientation of said first homologous vector sequence relative to said second homologous vector sequence is the same as the 5′-3′ orientation of said first region relative to said second region of said target sequence;    wherein the vector is capable of modifying said target DNA sequence by homologous recombination of said first homologous vector DNA sequence with said first region of said target sequence and of said second homologous vector DNA sequence with said second region of said target sequence.    
     
     
         101 . A transgenic mouse having a genome comprising a positive selection marker DNA sequence inserted into a non-selectable gene by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genomic DNA is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.  
     
     
         102 . The transgenic mouse of  claim 101  wherein said non-selectable gene is a gene of the immune system.  
     
     
         103 . The transgenic mouse of  claim 102 , wherein said genome further comprises a heterologous protein coding sequence other than a positive selection marker DNA sequence.  
     
     
         104 . The transgenic mouse of  claim 103 , wherein said heterologous protein coding sequence is a human gene.  
     
     
         105 . The transgenic mouse of  claim 104 , wherein said human gene is a gene of the human immune system.  
     
     
         106 . The transgenic mouse of  claim 105 , wherein said genome is free of a positive screening marker DNA sequence.  
     
     
         107 . The transgenic mouse of  claim 106 , wherein said genome is free of a bacterial tRNA suppressor gene.  
     
     
         108 . A transgenic mouse having a genome comprising a human gene replacing a corresponding mouse gene, wherein said human gene is inserted into said genome by a homologous recombination event between said genome and a targeting vector, without a duplication of genomic DNA sequence by said even and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event  
     
     
         109 . The transgenic mouse of  claim 108 , wherein said human gene is a gene of the immune system.  
     
     
         110 . An isolated cell having a genome comprising a positive selection marker DNA sequence inserted into a non-selectable gene by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.  
     
     
         111 . The cell of  claim 110 , selected from the group consisting of hematopoietic, epithelial, liver, lung, bone marrow, endothelial, mesenchymal, neural and muscle stem cells.  
     
     
         112 . The cell of  claim 111 , which is a cell of a vertebrate animal.  
     
     
         113 . The cell of  claim 112 , which is a human cell.  
     
     
         114 . The cell of  claim 111 , which is a bone marrow stem cell.  
     
     
         115 . The cell of  claim 112 , which is a simian cell.  
     
     
         116 . An isolated mouse embryonic stem cell having a genome comprising a positive selection marker DNA sequence inserted into a gene other than hprt by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.