Methods of modifying eukaryotic cells

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.

We claim,  
     
         1 . A method of replacing, in whole or in part, in a non-human eukaryotic cell, an endogenous immunoglobulin variable region gene locus with an homologous or orthologous human gene locus comprising: 
 a) obtaining a large cloned genomic fragment containing, in whole or in part, the homologous or orthologous human 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);    c) introducing the LTVEC of (b) into the eukaryotic cells to replace, 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 gene locus.    
     
     
         2 . The method of  claim 1  further comprising: 
 e) obtaining a large cloned genomic fragment containing a part of the homologous or orthologous human gene locus that differs from the fragment of (a);  
 f) using bacterial homologous recombination to genetically modify the cloned genomic fragment of (e) to create a second LTVEC;  
 g) introducing the second LTVEC of (f) into the eukaryotic cells identified in step (d) to replace, in whole or in part, the endogenous immunoglobulin variable gene locus; and  
 h) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (g) 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 gene locus.  
 
     
     
         3 . The method of  claim 2  wherein steps (e) through (h) are repeated until the endogenous immunoglobulin variable region gene locus is replaced in whole with an homologous or orthologous human gene locus.  
     
     
         4 . The method of  claim 1  wherein the immunoglobulin variable gene locus is a locus selected from the group consisting of: 
 a) a variable gene locus of the kappa light chain;  
 b) a variable gene locus of the lambda light chain; and  
 c) a variable gene locus of the heavy chain.  
 
     
     
         5 . The method of  claim 4  wherein the quantitative assay comprises quantitative PCR, FISH, comparative genomic hybridization, isothermic DNA amplification, or quantitative hybridization to an immobilized probe.  
     
     
         6 . The method of  claim 5  wherein the quantitative PCR comprises TaqMan® technology or quantitative PCR using molecular beacons.  
     
     
         7 . A method of replacing, in whole or in part, in a mouse embryonic stem cell, an endogenous immunoglobulin variable region gene locus with its homologous or orthologous human gene locus comprising: 
 a) obtaining a large cloned genomic fragment containing, in whole or in part, the homologous or orthologous human 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;    c) introducing the large targeting vector of (b) into mouse embryonic stem cells to replace, 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 gene locus.    
     
     
         8 . The method of  claim 7  further comprising: 
 e) obtaining a large cloned genomic fragment containing a part of the homologous or orthologous human gene locus that differs from the fragment of (a);  
 f) using bacterial homologous recombination to genetically modify the cloned genomic fragment of (e) to create a large targeting vector for use in the embryonic stem cells;  
 g) introducing the large targeting vector of (f) into the mouse embryonic stem cells identified in step (d) to replace, in whole or in part, the endogenous immunoglobulin variable gene locus; and  
 h) using a quantitative assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (g) to identify those mouse embryonic stem cells in which the endogenous immunoglobulin variable region gene locus has been replaced, in whole or in part, with the homologous or orthologous human gene locus.  
 
     
     
         9 . The method of  claim 8  wherein steps (e) through (h) are repeated until the endogenous immunoglobulin variable region gene locus is replaced in whole with an homologous or orthologous human gene locus.  
     
     
         10 . The method of  claim 7  wherein the immunoglobulin variable gene locus comprises a locus selected from the group consisting of 
 a) a variable gene locus of the kappa light chain;  
 b) a variable gene locus of the lambda light chain; and  
 c) a variable gene locus of the heavy chain.  
 
     
     
         11 . A genetically modified immunoglobulin variable region gene locus produced by the method of  claim 1 ,  4 ,  7 , or  10 .  
     
     
         12 . A genetically modified eukaryotic cell comprising a genetically modified immunoglobulin variable region gene locus produced by the method of  claim 1 ,  4 ,  7  or  10 .  
     
     
         13 . A non-human organism comprising a genetically modified immunoglobulin variable region gene locus produced by the method of  claim 1 ,  4 ,  7  or  10 .  
     
     
         14 . A mouse embryonic stem cell containing a genetically modified immunoglobulin variable region gene locus produced by the method of  claim 7  or  10 .  
     
     
         15 . An embryonic stem cell of  claim 14  wherein the mouse heavy chain variable region locus is replaced, in whole or in part, with a human heavy chain variable gene locus.  
     
     
         16 . An embryonic stem cell of  claim 14  wherein the mouse kappa light chain variable region locus is replaced, in whole or in part, with a human kappa light chain variable region locus.  
     
     
         17 . An embryonic stem cell of  claim 14  wherein the mouse lambda light chain variable region locus is replaced, in whole or in part, with a human lambda light chain variable region locus.  
     
     
         18 . An embryonic stem cell of  claim 14  wherein the heavy and light chain variable region gene loci are replaced, in whole, with their human homologs or orthologs.  
     
     
         19 . A mouse produced from the embryonic stem cell of  claim 15 .  
     
     
         20 . A mouse produced from the embryonic stem cell of  claim 16 .  
     
     
         21 . A mouse produced from the embryonic stem cell of  claim 17 .  
     
     
         22 . A mouse produced from the embryonic stem cell of  claim 18 .  
     
     
         23 . An antibody comprising a human variable region encoded by the genetically modified variable gene locus of  claim 11 .  
     
     
         24 . An antibody of  claim 23  further comprising a non-human constant region.  
     
     
         25 . An antibody of  claim 23  further comprising a human constant region.  
     
     
         26 . A transgenic mouse having a genome comprising entirely human heavy and light chain variable region loci operably linked to entirely endogenous mouse constant region loci such that the mouse produces a serum containing an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation.  
     
     
         27 . A transgenic mouse having a genome comprising human heavy and/or light chain variable region loci operably linked to endogenous mouse constant region loci such that the mouse produces a serum containing an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation.  
     
     
         28 . A transgenic mouse containing an endogenous immunoglobulin variable region locus that has been replaced with an homologous or orthologous human variable region locus, such mouse being produced by a method comprising: 
 a) obtaining one or more large cloned genomic fragments that, when combined, span the homologous or orthologous human variable region locus;    b) using bacterial homologous recombination to genetically modify the cloned genomic fragment(s) of (a) to create large targeting vector(s) for use in mouse embryonic stem cells;    c) introducing the large targeting vector(s) of (b) into mouse embryonic stem cells to replace the endogenous variable region locus in the cells;    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 region locus has been replaced with the homologous or orthologous human variable region locus;    e) introducing the mouse embryonic stem cell of (d) into a blastocyst; and    f) introducing the blastocyst of (e) into a surrogate mother for gestation.    
     
     
         29 . The transgenic mouse of  claim 27  or  28  wherein the immunoglobulin variable region gene locus comprises one or more loci selected from the group consisting of: 
 a) a variable gene locus of the kappa light chain;  
 b) a variable gene locus of the lambda light chain; and  
 c) a variable gene locus of the heavy chain.  
 
     
     
         30 . The method of  claim 7 ,  8 ,  9 , or  10  wherein the mouse embryonic stem cell is derived from a transgenic mouse produced by the method of  claim 28 .  
     
     
         31 . A method of making a human antibody comprising: 
 a) exposing the mouse of  claim 26  to antigenic stimulation, such that the mouse produces an antibody against the antigen;    b) isolating the DNA encoding the variable regions of the heavy and light chains of the antibody;    c) operably linking the DNA encoding the variable regions of (b) to DNA encoding the human heavy and light chain constant regions in a cell capable of expressing active antibodies;    d) growing the cell under such conditions as to express the human antibody; and    e) recovering the antibody.    
     
     
         32 . The method of  claim 31  wherein the cell is a CHO cell.  
     
     
         33 . The method of  claim 31  wherein said DNA of step (b) is isolated from a hybridoma created from the spleen of the mouse exposed to antigenic stimulation in step (a).  
     
     
         34 . The method of  claim 31  wherein said DNA is isolated by PCR.