Methods of modifying eukaryotic cells
First Claim
1. A method for genetically modifying an endogenous gene or chromosomal locus of interest in isolated eukaryotic cells, comprising:
- a) obtaining a large cloned genomic fragment greater than 20 kb containing a DNA sequence of interest;
b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC), such LTVEC having homology arms which total greater than 20 kb;
c) introducing the LTVEC of (b) into the isolated eukaryotic cells to modify by homologous recombination the endogenous gene or chromosomal locus in the cells; and
d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified.
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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.
197 Citations
14 Claims
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1. A method for genetically modifying an endogenous gene or chromosomal locus of interest in isolated eukaryotic cells, comprising:
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a) obtaining a large cloned genomic fragment greater than 20 kb containing a DNA sequence of interest;
b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC), such LTVEC having homology arms which total greater than 20 kb;
c) introducing the LTVEC of (b) into the isolated eukaryotic cells to modify by homologous recombination the endogenous gene or chromosomal locus in the cells; and
d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for genetically modifying an endogenous gene or chromosomal locus of interest in isolated mouse embryonic stem cells, comprising:
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a) obtaining a large cloned genomic fragment greater than 20 kb which contains a DNA sequence of interest, wherein the large cloned DNA fragment is homologous to the endogenous gene or chromosomal locus;
b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the mouse embryonic stem cells, the large targeting vector having homology arms that total greater than 20 kb and wherein the genetic modification is deletion of a coding sequence, gene segment, or regulatory element;
c) introducing the large targeting vector of (b) into the mouse embryonic stem cells to modify by homologous recombination the endogenous gene or chromosomal locus in the cells; and
d) using a quantitative assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (c) to identify those mouse embryonic stem cells in which the endogenous gene or chromosomal locus has been genetically modified, wherein the quantitative assay is quantitative PCR.
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Specification