Patent ID: 8759105

Claim:
A method for genetically modifying a mouse embryonic stem (ES) cell, comprising: (a) targeting a mouse ES cell by homologous recombination with a targeting vector that comprises a human cloned genomic fragment larger than 20 kb containing a DNA sequence of interest that is orthologous to an endogenous mouse allele; (b) using a quantitative assay that detects modification of allele (MOA) in the mouse ES cell of (a) to identify a mouse ES cell in which the endogenous mouse allele has been genetically modified, the assay comprising: (i) exposing the DNA of the mouse ES cell of (a) with a first probe and a second probe, wherein the first probe binds within the endogenous mouse allele but not within the DNA sequences of interest, and the second probe binds within a reference gene but not within the endogenous mouse allele and not within the DNA sequence of interest, and wherein both probes generate a detectable signal upon binding, and wherein the reference gene is of known copy number; (ii) detecting the signal from the binding of the first probe and the signal from the binding of the second probe; and, (iii) comparing the signal from the binding of the second probe to the signal from the binding of the first probe, and determining from the comparison a copy number of the endogenous mouse allele; wherein the modification comprises replacing the endogenous mouse allele with the orthologous DNA sequence of interest.