Methods of Modifying Eurakyotic Cells

• US 20130130388A1
• Filed: 12/19/2012
• Published: 05/23/2013
• Est. Priority Date: 10/31/2000
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Murphy, Andrew J., Yancopoulos, George D.
• Granted Patent: US 10,227,625 B2
• Time in Patent Office: Days
• US Class Current: 435/463
• 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 targeted insertion of non-endogenous DNA at a desired position in a non-human genome of a eukaryotic cell, comprising

selecting a bacterial artificial chromosome (BAC) clone comprising a large genomic DNA fragment comprising a DNA sequence of interest;

employing the BAC clone to generate a large targeting vector comprising the large genomic DNA fragment comprising the DNA sequence of interest;

employing the large targeting vector to modify a non-human genome at a desired position with the large targeting vector.

2. The method of claim 1, wherein the non-endogenous DNA is homologous to DNA in the non-human genome at the desired position in the non-human genome.

3. The method of claim 1, wherein the non-endogenous DNA is orthologous to DNA in the non-human genome at the desired position in the non-human genome.

4. The method of claim 1, wherein the non-endogenous DNA comprises unrearranged human immunoglobulin sequences and the desired position is a non-human immunoglobulin locus.

5. The method of claim 1, wherein the non-human genome is a rodent genome.

6. The method of claim 5, wherein the rodent genome is a mouse genome.

7. The method of claim 1, wherein the eukaryotic cell is a mammalian cell.

8. The method of claim 1, wherein the mammalian cell is a rodent cell.

9. The method of claim 8, wherein the rodent cell is a mouse cell.

10. The method of claim 8, wherein the rodent cell is an embryonic stem cell.

11. The method of claim 10, wherein the rodent embryonic stem cell is a mouse embryonic stem cell.

12. The method of claim 1, wherein the large targeting vector comprising the genomic DNA fragment comprising the DNA sequence of interest comprises a modification selected from a point mutation in the DNA sequence of interest and a deletion in the DNA sequence of interest.

13. The method of claim 1, wherein the method is employed to modify the non-human genome of the eukaryotic cell so as to create a knockout, a knockin, a point mutation, an insertion, or a deletion in the non-human genome.

14. The method of claim 13, wherein the method is employed to create a knockin with a homologous or orthologous non-endogenous DNA in the non-human genome of the eukaryotic cell.

15. The method of claim 14, wherein the homologous or orthologous non-endogenous DNA is human DNA and the eukaryotic cell is a rodent cell.

16. The method of claim 15, wherein the rodent cell is a mouse cell.

17. The method of claim 16, wherein the mouse cell is a mouse embryonic stem cell.

18. A non-human genome of a mammalian cell comprising a targeted insertion of a large piece of non-endogenous DNA greater than 20 kb at a desired position in the non-human genome, wherein the non-endogenous DNA is homologous or orthologous to DNA in the non-human genome of the eukaryotic cell at the desired position.

19. The non-human genome of claim 18, wherein the mammalian cell is a rodent cell and the homologous or orthologous DNA in the rodent genome at the desired position is human DNA.

20. The non-human genome of claim 19, wherein the rodent cell is a mouse embryonic stem cell.