Method for generating hypermutable organisms

Abstract

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. The enhanced rate of mutation can be further augmented using mutagens. Moreover, the hypermutability of mismatch repair deficient cells can be remedied to stabilize cells or mammals with useful mutations.

1 . A method for generating a mutation in a gene of interest in a cell comprising the steps of:
 growing a cell comprising (a) a gene of interest and (b) a polynucleotide encoding a polypeptide comprising the N-terminal 133 amino acids of PMS2, said polynucleotide operably linked to a promoter, wherein said polypeptide is capable of inhibiting mismatch repair;   testing the cell to determine whether the gene of interest harbors a mutation that results in a detectable phenotype; and   restoring mismatch repair activity to said cell.   
     
     
         2 . The method of  claim 1  wherein said polynucleotide encodes PMS2. 
     
     
         3 . The method of  claim 1  wherein said polynucleotide encodes PMS2-134. 
     
     
         4 . The method of  claim 1  wherein said cells are prokaryotic cells. 
     
     
         5 . The method of  claim 1  wherein said cells are eukaryotic cells. 
     
     
         6 . The method of  claim 5  wherein said eukaryotic cells are mammalian cells. 
     
     
         7 . The method of  claim 1  further comprising the step of treating said cell with a mutagen. 
     
     
         8 . The cell produced according to the method of  claim 1 . 
     
     
         9 . A method for generating a library of genetically stable, hypermutated cells comprising:
 expressing in cells a polynucleotide encoding a polypeptide comprising the N-terminal 133 amino acids of PMS2, wherein said expression inhibits mismatch repair of said cells, and   restoring mismatch repair activity to said cells,   thereby producing a library of genetically stable, hypermutated cells.   
     
     
         10 . The method of  claim 9  wherein said polynucleotide encodes PMS2. 
     
     
         11 . The method of  claim 9  wherein said polynucleotide encodes PMS2-134. 
     
     
         12 . The method of  claim 9  wherein said cells are prokaryotic cells. 
     
     
         13 . The method of  claim 9  wherein said cells are eukaryotic cells. 
     
     
         14 . The method of  claim 13  wherein said eukaryotic cells are mammalian cells. 
     
     
         15 . The method of  claim 9  further comprising the step of treating said cells with a mutagen. 
     
     
         16 . A method for generating a genetically altered cell line comprising:
 introducing a polynucleotide encoding a polypeptide comprising the N-terminal 133 amino acids of PMS2, said polynucleotide operably linked to a promoter, wherein said polypeptide is capable of inhibiting mismatch repair, in a population of cells in culture, thereby inhibiting mismatch repair in said cells,   separating said population into individual members of the population,   identifying members of the population comprising a mutation in a gene of interest,   restoring mismatch repair activity to said members of the population comprising a mutation in the gene of interest, and   expanding said members comprising a mutation in the gene of interest,   thereby generating a genetically altered cell line.   
     
     
         17 . The method of  claim 16  wherein said polynucleotide encodes PMS2. 
     
     
         18 . The method of  claim 16  wherein said polynucleotide encodes PMS2-134. 
     
     
         19 . The method of  claim 16  wherein said cells are prokaryotic cells. 
     
     
         20 . The method of  claim 16  wherein said cells are eukaryotic cells. 
     
     
         21 . The method of  claim 20  wherein said eukaryotic cells are mammalian cells. 
     
     
         22 . The method of  claim 16  further comprising the step of treating said cell with a mutagen. 
     
     
         23 . The genetically altered cell line produced according to the method of  claim 16 .