Patent ID: 7105348

Claim:
A method of creating, in an isolated mouse embryonic stem (ES) cell, a genetically modified endogenous gene locus flanked downstream by a site-specific recombination site comprising: (a) using bacterial homologous recombination to genetically modify a cloned genomic fragment of an endogenous gene locus to create a large targeting vector for use in eukaryotic cells (LTVEC), said LTVEC comprising a site-specific recombination site, a downstream homology arm containing a region homologous to the 3′ end of the endogenous gene locus region and an upstream homology arm within the locus, wherein the homology arms are larger than 20 kb and the site-specific recombination site is selected from one or more of loxP, lox511, and lox2272; (b) introducing the LTVEC of (a) into an isolated mouse ES cell; and (d) using a quantitative assay with a probe directed to an unmodified allele of the endogenous gene locus to detect reduced copy number of the unmodified allele compared to that of a reference gene in the cell from (b) thereby indicating modification of allele (MOA) in the endogenous gene locus of the cell, wherein the endogenous gene locus is flanked downstream by the site-specific recombination site.