Methods of Modifying Eukaryotic Cells
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.
1 . A transgenic mouse having a genome comprising a modified immunoglobulin heavy chain locus in which an endogenous heavy chain variable region has been replaced with an unrearranged human heavy chain variable region such that the unrearranged human heavy chain variable region is operably linked to an endogenous mouse heavy chain constant region.
2 . The mouse of claim 1 , wherein the human heavy chain variable region is contained on a human genomic DNA fragment that is larger than 20 kb.
3 . The mouse of claim 1 , wherein the human heavy chain variable region is contained on a human genomic DNA fragment that is larger than 100 kb.
4 . The mouse of claim 1 , wherein the genome further comprises a modified immunoglobulin light chain locus in which an endogenous light chain variable region has been replaced with an unrearranged human light chain variable region such that the unrearranged light chain variable region is operably linked to an endogenous mouse light chain constant region.
5 . The mouse of claim 4 , wherein the light chain immunoglobulin locus is a kappa light chain locus.
6 . The mouse of claim 4 , wherein the human heavy chain variable region is contained on a human genomic DNA fragment that is larger than 20 kb and the human light chain variable region is contained on a human genomic DNA fragment that is larger than 20 kb.
7 . The mouse of claim 4 , wherein the human heavy chain variable region is contained on a human genomic DNA fragment that is larger than 100 kb and the human light chain variable region is contained on a human genomic DNA fragment that is larger than 100 kb.
8 . The mouse of claim 1 , wherein the human unrearranged heavy chain variable region is capable of rearranging to form a functional heavy chain variable region gene.
9 . The mouse of claim 8 , wherein following rearrangement the mouse expresses an antibody comprising an antibody heavy chain variable region encoded by the functional heavy chain region gene.
10 . The mouse of claim 1 , wherein the mouse produces an antibody that comprises a human heavy chain variable region and a mouse heavy chain constant region.
11 . The mouse of claim 1 , wherein the mouse does not comprise a human immunoglobulin heavy chain constant gene.
12 . The mouse of claim 4 , wherein the mouse produces an antibody that comprises a human heavy chain variable region and a mouse heavy chain constant region, a human light chain variable region and a mouse light chain constant region.
13 . The mouse of claim 1 , wherein the modified immunoglobulin heavy chain locus comprises a murine heavy chain intronic enhancer, Em.
14 . The mouse of claim 1 , wherein the modified immunoglobulin heavy chain locus comprises a murine immunoglobulin 3′ enhancer region.
15 . A transgenic mouse having a genome comprising:
a. a modified immunoglobulin heavy chain locus in which an endogenous heavy chain variable region has been replaced with an unrearranged human heavy chain variable region such that the unrearranged human heavy chain variable region is operably linked to an endogenous mouse heavy chain constant region;
b. a modified immunoglobulin kappa light chain locus in which an endogenous kappa light chain variable region has been replaced with an unrearranged human kappa light chain variable region such that the unrearranged kappa light chain variable region is operably linked to an endogenous mouse light chain constant region; and
c. a modified immunoglobulin lambda light chain locus in which an endogenous lambda light chain variable region has been replaced with an unrearranged human lambda light chain variable region such that the unrearranged lambda light chain variable region is operably linked to an endogenous mouse lambda light chain constant region.
16 . The mouse of claim 15 , wherein the human heavy chain variable region is contained on a human genomic DNA fragment that is larger than 20 kb, the human kappa light chain variable region is contained on a human genomic DNA fragment that is larger than 20 kb, and the human lambda light chain variable region is contained on a human genomic DNA fragment that is larger than 20 kb.
17 . The mouse of claim 15 , wherein the human heavy chain variable region is contained on a human genomic DNA fragment that is larger than 100 kb, the human kappa light chain variable region is contained on a human genomic DNA fragment that is larger than 100 kb, and the human lambda light chain variable region is contained on a human genomic DNA fragment that is larger than 100 kb.
18 . The mouse of claim 15 , wherein the human unrearranged heavy chain variable region is capable of rearranging to form a functional heavy chain variable region gene, the human unrearranged kappa light chain variable region is capable of rearranging to form a functional kappa light chain variable region gene, and the human unrearranged lambda light chain variable region is capable of rearranging to form a functional lambda light chain variable region gene.
19 . The mouse of claim 18 , wherein the mouse expresses an antibody having a heavy chain comprising a heavy chain variable region encoded by the functional heavy chain variable region gene and a light chain comprising a light chain variable region encoded by the functional kappa light chain variable region gene or the functional lambda light chain variable region gene.
20 . The mouse of claim 15 , wherein the mouse produces an antibody that has a heavy chain that comprises a human heavy chain variable region and a mouse heavy chain constant region and has a light chain that comprises a human light chain variable region and a mouse light chain constant region.
21 . The mouse of claim 15 , wherein the mouse does comprises neither a human immunoglobulin heavy chain constant gene nor a human immunoglobulin light chain constant gene.
22 . The mouse of claim 15 , wherein the mouse is homozygous for the modified immunoglobulin heavy chain locus, the modified immunoglobulin kappa light chain locus and the modified immunoglobulin lambda light chain locus.
23 . The mouse of claim 15 , wherein the modified immunoglobulin heavy chain locus comprises a murine heavy chain intronic enhancer, Em.
24 . The mouse of claim 15 , wherein the modified immunoglobulin heavy chain locus comprises a murine immunoglobulin 3′ enhancer region.