Patent ID: 6916611

Claim:
A method for determimng efficiency of sequence-specific modification of intracellular DNA by homologous replacement in the nucleus of eukaryotic cells, the method comprising: (a) co-transfecting eukaryotic cells with: (i) an expression vector comprising a mutant marker gene and a second marker gene; and (ii) a gene targeting element consisting of sufficient wild type gene sequence to correct by homologous replacement the mutant marker gene set forth in (i) and lacking expression control sequence; (b) isolating a nuclear fraction from the transfected cells; (c) assaying the nuclear fraction for the presence of wild type marker gene and the second marker gene, wherein the assaying comprises: (i) extracting the vector from the nuclear fraction; (ii) transforming bacteria with the extracted vector; and (iii) growing the transformed bacteria under conditions sufficient to reveal presence of the wild type marker gene and the second marker gene; and (d) determining; the proportion of corrected sequences, whereby the number of bacteria in which the wild type marker gene is present is divided by the number of bacteria in which the second marker gene is present; the proportion present in the nuclear fraction being indicative of efficiency of sequence-specific modification of intracellular DNA by homologous replacement in the nucleus, whereby a higher proportion indicates more efficient replacement.