Methods of modifying eukaryotic cells

• US 10,227,625 B2
• Filed: 12/19/2012
• Issued: 03/12/2019
• Est. Priority Date: 10/31/2000
• Status: Expired due to Fees

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Murphy, Andrew J., Yancopoulos, George D., Karow, Margaret, Macdonald, Lynn, Stevens, Sean, Economides, Aris N., Valenzuela, David M.
• Primary Examiner(s): Singh, Anoop K
• Assistant Examiner(s): Sgagias, Magdalene K
• Application Number:
  US13/719,842
• Publication Number:
  US 20130130388A1
• Time in Patent Office: 2,274 Days
• 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 method for genetically modifying an endogenous immunoglobulin heavy chain variable region locus in isolated mouse embryonic stern (ES) cells, comprising;

(a) using bacterial homologous recombination to genetically modify a cloned genomic fragment of an endogenous mouse immunoglobulin variable region gene locus to create a first large targeting vector for use in eukaryotic cells (LTVEC), said LTVEC comprising a site-specific recombination site, and a human insert spanning from several V gene segments through the entire DJ region, which are flanked by a downstream homology arm containing a region adjacent to but not including the mouse J segments and an upstream homology arm within the locus, wherein the homology arms are larger than 20 kb and the site-specific recombination site is selected from one or more of loxP and lox511;

(b) using bacterial homologous recombination to genetically modify a cloned genomic fragment of an endogenous mouse immunoglobulin variable region gene locus to create a second LTVEC comprising a site-specific recombination site, an upstream homology arm containing a region that flanks the 5′

end of the endogenous mouse immunoglobulin variable region gene locus and a downstream homology arm within the locus;

(c) introducing the first and second LTVECs into an isolated mouse ES cell;

(d) using a quantitative assay with a probe directed to an unmodified allele of the endogenous immunoglobulin variable region gene locus to detect reduced copy number of the unmodified allele compared to that of a reference gene in the mouse ES cell from (c) thereby indicating modification of allele (MOA) in the endogenous gene locus of the mouse ES cell, wherein the endogenous mouse gene locus is flanked by the site-specific recombination sites of step (a) and step (b); and

(e) introducing a recombinase into the mouse ES cell identified in step (d), wherein the entire endogenous mouse immunoglobulin variable region gene locus flanked by the site-specific recombination sites is deleted, wherein the human insert spanning from several V gene segments through the entire DJ region replaces the endogenous mouse immunoglobulin variable region locus, such that human variable region gene segments in the human insert are operably linked to the entirely endogenous mouse constant region.

2. The method of claim 1, wherein the genomic fragment is larger than 100 kb.

3. The method of claim 1, wherein the endogenous mouse immunoglobulin enhancer Eμ

remains intact upstream of the endogenous mouse immunogiobulin heavy chain constant region gene.