Compositions and Methods for Modifying Cells
First Claim
1. A method for detecting the replacement of an endogenous allele with a modified allele in a cell, the method comprising:
- (a) exposing the DNA of the cell of with 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, and 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.
29 Citations
37 Claims
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1. A method for detecting the replacement of an endogenous allele with a modified allele in a cell, the method comprising:
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(a) exposing the DNA of the cell of with 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, and 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, 12, 13, 14, 15, 16)
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17. A method for replacing an endogenous allele with a modified allele in a cell, the method comprising:
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(a) targeting the cell by homologous recombination with a targeting vector that comprises a DNA sequence of the modified allele; (b) exposing the DNA of the cell of with 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, and wherein both probes generate a detectable signal upon binding, and wherein the reference gene is of known copy number; (c) detecting the signal from the binding of the first probe and the signal from the binding of the second probe; and (d) 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 (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A composition comprising:
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(a) a cell comprising an allele in which an endogenous allele has been replaced with a modified allele comprising an exogenous nucleic acid sequence; (b) a first detectable nucleic acid probe that selectively binds within the endogenous allele but not within the modified allele; (c) a second detectable nucleic acid probe that selectively binds within a reference nucleic acid sequence of known copy number within the genome of the cell, wherein the second detectable nucleic acid probe does not bind within the endogenous allele or the modified allele. - View Dependent Claims (34, 35, 36, 37)
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Specification