Methods of modifying eukaryotic cells

• US 20070061900A1
• Filed: 11/09/2006
• Published: 03/15/2007
• Est. Priority Date: 10/31/2000
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Yancopoulos, George, Murphy, Andrew
• Granted Patent: US 8,791,323 B2
• Time in Patent Office: Days
• US Class Current: 800/6
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/204 : of bacterial origin, e.g. BAC
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00

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.

Claims

1. A transgenic mouse having a genome comprising human heavy and light chain immunoglobulin variable region gene loci, wherein the human heavy and light chain variable region loci replace mouse endogenous heavy and light chain immunoglobulin variable region gene loci, and the human heavy and light chain variable region loci are linked to endogenous mouse heavy and light chain immunoglobulin constant region gene loci to form hybrid loci, whereby the hybrid loci rearrange during B-cell development such that the mouse produces a serum containing an antibody comprising human heavy and light chain immunoglobulin variable regions and mouse heavy and light chain immunoglobulin constant regions in response to antigenic stimulation.

2. The transgenic mouse of claim 1 wherein the endogenous light chain immunoglobulin constant region gene locus is a kappa light chain.

3. The transgenic mouse of claim 1, wherein the endogenous light chain immunoglobulin constant region is a lambda light chain region.

4. The transgenic mouse of claim 1, wherein the mouse has heavy and light chain immunoglobulin variable region loci that are entirely human linked to heavy and light chain immunoglobulin constant region loci that are entirely mouse to form the hybrid loci.

5. A method of producing an antibody, comprising

providing a transgenic mouse having a genome comprising human heavy and light chain immunoglobulin variable region gene loci, wherein the human heavy and light chain variable region loci replace mouse endogenous heavy and light chain immunoglobulin variable region gene loci, and the human heavy and light chain variable region loci are linked to endogenous mouse heavy and light chain immunoglobulin constant region gene loci to form hybrid loci, whereby the hybrid loci rearrange during B-cell development such that the mouse produces a serum containing an antibody comprising human heavy and light chain immunoglobulin variable regions and mouse heavy and light chain immunoglobulin constant regions in response to antigenic stimulation

stimulating the mouse with an antigen;

preparing a hybridoma expressing the antibody from the mouse.

6. The method of claim 5, further comprising preparing a human antibody comprising the human heavy and light chain immunoglobulin variable regions.

7. The method of claim 6, wherein the human antibody further comprises human heavy and light chain constant regions.

8. A method of producing an antibody, comprising

providing a transgenic mouse having a genome comprising human heavy and light chain immunoglobulin variable region gene loci, wherein the human heavy and light chain variable region loci replace mouse endogenous heavy and light chain immunoglobulin variable region gene loci, and the human heavy and light chain variable region loci are linked to endogenous mouse heavy and light chain immunoglobulin constant region gene loci to form hybrid loci, whereby the hybrid loci rearrange during B-cell development such that the mouse produces a serum containing an antibody comprising human heavy and light chain immunoglobulin variable regions and mouse heavy and light chain immunoglobulin constant regions in response to antigenic stimulation

stimulating the mouse with an antigen;

isolating DNA encoding the heavy and light chain variable regions of the antibody from the mouse;

operably linking the DNA encoding the variable regions to DNA encoding human heavy and light chain constant regions

expressing a human antibody comprising the human heavy and light chain variable regions and the human heavy and light chain constant regions.