Methods of modifying eukaryotic cells
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsAndrew J. Murphy, George D. Yancopoulos
Life of this patent
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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.
Claims17
A method of replacing, in whole or in part, in an isolated non-human eukaryotic cell, an endogenous immunoglobulin variable region gene locus with an homologous or orthologous human immunoglobulin variable gene locus comprising:
- a) obtaining a large cloned genomic fragment greater than 20 kb containing, in whole or in part, the homologous or orthologous human immunoglobulin variable gene locus;
- b) using bacterial homologous recombination to genetically modify the cloned genomic fragment of (a) to create a large targeting vector for use in the eukaryotic cells (LTVEC), the LTVEC having homology arms totaling greater than 20 kb;
- c) introducing the LTVEC of (b) into the isolated eukaryotic cells to replace by homologous recombination, in whole or in part, the endogenous immunoglobulin variable gene locus;
- 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 immunoglobulin variable region gene locus has been replaced, in whole or in part, with the homologous or orthologous human immunoglobulin variable gene locus.
A method of replacing, in whole or in part, in an isolated mouse embryonic stem cell, an endogenous immunoglobulin variable region gene locus with the homologous or orthologous human immunoglobulin variable gene locus comprising:
- a) obtaining a large cloned genomic fragment greater than 20 kb containing, in whole or in part, the homologous or orthologous human immunoglobulin variable gene 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 embryonic stem cells, the large targeting vector having homology arms greater than 20 kb;
- c) introducing the large targeting vector of (b) into isolated mouse embryonic stem cells to replace by homologous recombination, in whole or in part, the endogenous immunoglobulin variable gene locus in the cells;
- and
- d) using a quantitative PCR 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 variable gene locus has been replaced, in whole or in part, with the homologous or orthologous human immunoglobulin variable gene locus.
Dependencies are inferred from the claim text.