Methods of Modifying Eukaryotic Cells

• US 20110258710A1
• Filed: 06/07/2011
• Published: 10/20/2011
• Est. Priority Date: 10/31/2000
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: YANCOPOULOS, GEORGE D., MURPHY, ANDREW J.
• Granted Patent: US 9,376,699 B2
• Time in Patent Office: Days
• US Class Current: 800/4
• 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

(a) a hybrid human variable/mouse constant immunoglobulin heavy chain locus comprising a replacement of mouse immunoglobulin heavy chain V, D, and J gene segments with human heavy chain immunoglobulin V, D, and J gene segments, wherein the replacement is at a mouse immunoglobulin heavy chain locus, and wherein the human V, D, and J gene segments are linked to an endogenous mouse immunoglobulin heavy chain constant region gene sequence;

(b) a hybrid human variable/mouse constant immunoglobulin light chain locus comprising a replacement of mouse immunoglobulin light chain V and J gene segments with human light chain immunoglobulin V and J gene segments, wherein the replacement is at a mouse immunoglobulin light chain locus, and wherein the human V and J gene segments are linked to an endogenous mouse immunoglobulin light chain constant region gene sequence;

(c) a functional murine immunoglobulin heavy chain intronic enhancer (Em) at an endogenous murine immunoglobulin heavy chain locus, wherein the Em is functionally able to participate in recombination of the human heavy chain V, D, and J gene segments; and

,

(d) a functional murine immuloglobulin heavy chain 3′

enhancer region at an endogenous murine immunoglobulin heavy chain locus, wherein the functional murine immunoglobulin heavy chain 3′

enhancer regions is functionally able to participate in one or more of class switching and heavy chain gene expression during B cell differentiation;

wherein the mouse expresses a chimeric human variable/mouse constant antibody, and the mouse is incapable of forming a chimeric human variable/mouse constant antibody gene by trans-splicing or trans-rearrangements.

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 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 hybrid loci.

4. A method of producing an antibody, comprising:

providing a genetically modified mouse, wherein the genetic modification is in the germline of the mouse, and comprises

(a) a replacement of mouse immunoglobulin heavy chain V, D, and J gene segments with human heavy chain immunoglobulin V, D, and J gene segments, wherein the replacement is at a mouse immunoglobulin heavy chain locus, and wherein the human V, D, and J gene segments are linked to an endogenous mouse immunoglobulin heavy chain constant region gene sequence, and,

(b) a replacement of mouse immunoglobulin light chain V and J gene segments with human light chain immunoglobulin V and J gene segments, wherein the replacement is at a mouse immunoglobulin light chain locus, and wherein the human V and J gene segments are linked to an endogenous mouse immunoglobulin light chain constant region gene sequence;

wherein the replacement of (a) forms a hybrid heavy chain locus, the replacement of (b) forms a hybrid light chain locus, and the hybrid heavy chain locus and the hybrid light chain locus are rearrange during B cell development such that the mouse produces a serum containing an antibody encoded by an antibody gene comprising human heavy and light chain immunoglobulin variable regions and mouse heavy and light chain immunoglobulin constant regions in response to antigenic stimulation, wherein the antibody gene is not produced through trans-splicing or trans-rearrangements;

stimulating the mouse with antigen;

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

operably linking nucleic acids encoding the variable regions to nucleic acids 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.

5. The mouse of claim 4, comprising a functional murine immunoglobulin heavy chain intronic enhancer (Em) at an endogenous murine immunoglobulin heavy chain locus, wherein the Em is functionally able to participate in recombination of the human heavy chain V, D, J gene segments.

6. The mouse of claim 5, comprising a functional murine immunoglobulin heavy chain 3′

enhancer region at an endogenous murine immunoglobulin heavy chain locus, wherein the functional murine immunoglobulin heavy chain 3′

enhancer regions is functionally able to participate in one or more of class switching and heavy chain gene expression during B cell differentiation.

7. A method for replacing an endogenous mouse immunoglobulin variable region gene locus with a human immunoglobulin variable region gene locus, comprising:

(a) obtaining a large cloned genomic fragment containing the homologous or orthologous human gene locus in whole or in part;

(b) using bacterial homologous recombination to genetically modify the cloned fragment of (a) to create a large targeting vector for use in a mouse ES cell;

(c) introducing the large targeting vector into the mouse ES cell to replace, in whole or in part, the endogenous mouse immunoglobulin variable region gene locus;

(d) using a modification of allele assay to detect to detect modification of the endogenous mouse immunoglobulin variable region gene locus, wherein the modification of allele assay detects the modification of one endogenous mouse immunoglobulin variable region allele to identify cells that have undergone homologous recombination at an endogenous mouse immunoglobulin variable region gene locus.

8. The method of claim 7, wherein the modification of allele assay is a quantitative assay that does not require the use of probes located outside the endogenous mouse immunoglobulin variable region gene locus that is modified.

9. The method of claim 7, wherein the modification of allele assay is a quantitative assay that measures reduction in copy number of an unmodified endogenous mouse immunoglobulin variable region gene locus.

10. The method of claim 7, wherein the modification of allele assay quantitates a number of alleles that have undergone homologous recombination.