Methods of modifying eukaryotic cells

• US 8,502,018 B2
• Filed: 06/20/2011
• Issued: 08/06/2013
• Est. Priority Date: 10/31/2000
• Status: Expired due to Term

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Murphy, Andrew J., Yancopoulos, George D.
• Primary Examiner(s): Leffers, Jr., Gerald
• Assistant Examiner(s): SGAGIAS, MAGDALENE K
• Application Number:
  US13/164,176
• Publication Number:
  US 20110283376A1
• Time in Patent Office: 778 Days
• Field of Search: 800/18, 800/8, 800/21, 800/22, 435/325, 435/320.1
• US Class Current: 800/18
• 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 genetically modified mouse, comprising in its germline human unrearranged variable region gene segments inserted at an endogenous mouse immunoglobulin locus.

2. The mouse of claim 1, wherein the human unrearranged variable region gene segments are heavy chain gene segments, and the mouse immunoglobulin locus is a heavy chain locus.

3. The mouse of claim 1, wherein the human unrearranged variable region gene segments are light chain gene segments, and the mouse immunoglobulin locus is a light chain locus.

4. The mouse of claim 3, wherein the light chain gene segments are human kappa light chain gene segments.

5. The mouse of claim 1, wherein the unrearranged variable region gene segments are contained on a genomic DNA fragment larger than 20 kb.

6. The mouse of claim 1, wherein the human variable region gene segments are capable of rearranging to form a functional V region gene.

7. The mouse of claim 1, where following rearrangement the mouse expresses a functional antigen-binding molecule encoded by the human gene segments.

8. The mouse of claim 1, wherein rearrangement of the human variable gene segments in the mouse results in a variable region gene that comprises a rearranged human variable region gene linked to a mouse constant region gene.

9. The mouse of claim 1, wherein the mouse produces an antibody that comprises a human variable region and a mouse constant region.

10. The mouse of claim 1, wherein the mouse does not comprise a human immunoglobulin constant gene.

11. A genetically modified mouse, comprising in its germline human unrearranged variable region gene segments linked to a mouse constant region gene, wherein the mouse lacks a human constant region gene, and wherein the mouse constant region gene is at an endogenous mouse immunoglobulin locus.

12. The mouse of claim 11, wherein the human unrearranged variable region gene segments are heavy chain gene segments.

13. The mouse of claim 11, wherein the human unrearranged variable region gene segments are light chain gene segments.

14. The mouse of claim 13, wherein the light chain gene segments are human kappa light chain gene segments.

15. The mouse of claim 11, wherein the unrearranged variable region gene segments are contained on a genomic DNA fragment larger than 20 kb.

16. The mouse of claim 11, wherein the human variable region gene segments are capable of rearranging to form a functional variable region gene.

17. The mouse of claim 11, where following rearrangement the mouse expresses a functional antigen-binding molecule encoded by the human gene segments.

18. The mouse of claim 11, wherein rearrangement of the human variable gene segments in the mouse results in a variable region gene that comprises a rearranged human variable region gene operably linked to a mouse constant region gene.

19. The mouse of claim 11, wherein the mouse produces an antibody that comprises a human variable region and a mouse constant region.

20. A mouse, comprising a modification in the germline of the mouse, wherein the modification comprises

(a) a hybrid heavy chain locus comprising an insertion of human immunoglobulin heavy chain V, D, and J gene segments, wherein the human heavy chain immunoglobulin V, D, and J gene segments are linked to a mouse immunoglobulin heavy chain gene, wherein the mouse immunoglobulin heavy chain gene is at an endogenous mouse immunoglobulin locus;

(b) a hybrid light chain locus comprising an insertion of human immunoglobulin light chain V and J gene segments, wherein the human V and J gene segments are linked to a mouse immunoglobulin light chain constant region gene sequence;

wherein (a) rearranges to form a hybrid heavy chain sequence comprising a human variable region linked to a mouse constant region, and (b) rearranges to form a hybrid light chain sequence comprising a human variable region linked to a mouse constant region, and wherein the mouse is incapable of forming an antibody that comprises a human variable region and a human constant region.