Patent ID: 6069010
Filing Date: 2000-05-30
Classification: C12N

Abstract:
A recombination based method for the generation of a specific disruption in a cloned mammalian genomic sequence of interest, the method comprising:contacting in a bacterial cell,(A) a first vector, containing (i) a mammalian genomic DNA sequence of interest of at least about 25 kb in length, (ii) a cleavage site for an endonuclease that cuts once in the vector backbone, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, and(B) a second vector containing (I) a recombination sequence of from about 20 to 1000 bp and having sequence identity with a portion of said genomic DNA sequence of interest, (ii) a positive selection marker for mammalian cells, (iii) a positive selection marker for said bacterial cell, and (iv) a single copy number origin of replication functional in said bacterial cell, that is disparate and compatible with said origin of replication in said first vector andmaintaining said bacterial cell under conditions that promote homologous recombination; wherein said second vector integrates into said first vector by homologous recombination between said mammalian genomic DNA sequence and said recombination sequence to provide a single recombinant vector comprising an insertion in said mammalian genomic sequence of interest, which recombinant vector is stable and maintained as a single copy in said bacterial cell.