Method for detecting the replacement of an endogenous allele with a modified allele by homologous recombination in a mouse ES cell
First Claim
1. A method for detecting the replacement of an endogenous allele with a modified allele by homologous recombination in a mouse embryonic stem (ES) cell, the method comprising:
- (a) exposing the DNA of the mouse ES cell to a first probe and a second probe, wherein the first probe binds within the endogenous allele but not within the modified allele, and the second probe binds within a reference gene but not within the endogenous allele and not within the modified allele, wherein both probes generate a detectable signal upon binding, and wherein the reference gene is of known copy number;
(b) detecting the signal from the binding of the first probe and the signal from the binding of the second probe; and
(c) 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 allele.
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Abstract
A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.
15 Citations
11 Claims
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1. A method for detecting the replacement of an endogenous allele with a modified allele by homologous recombination in a mouse embryonic stem (ES) cell, the method comprising:
- (a) exposing the DNA of the mouse ES cell to a first probe and a second probe, wherein the first probe binds within the endogenous allele but not within the modified allele, and the second probe binds within a reference gene but not within the endogenous allele and not within the modified allele, wherein both probes generate a detectable signal upon binding, and wherein the reference gene is of known copy number;
(b) detecting the signal from the binding of the first probe and the signal from the binding of the second probe; and
(c) 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 allele. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- (a) exposing the DNA of the mouse ES cell to a first probe and a second probe, wherein the first probe binds within the endogenous allele but not within the modified allele, and the second probe binds within a reference gene but not within the endogenous allele and not within the modified allele, wherein both probes generate a detectable signal upon binding, and wherein the reference gene is of known copy number;
Specification