Methods of Modifying Eukaryotic Cells

• US 20130137101A1
• Filed: 12/19/2012
• Published: 05/30/2013
• Est. Priority Date: 10/31/2000
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Economides, Aris N., Murphy, Andrew J., Valenzuela, David M., Frendewey, David, Yancopoulos, George D.
• Granted Patent: US 8,846,402 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2810/10 Vectors comprising a non-peptidic targeting moiety
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

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 targeting a large cloned genomic fragment to a desired position in a non-human genome, comprising

(a) using bacterial homologous recombination to precisely engineer a genetic modification within a large cloned genomic fragment to form a large targeting vector, wherein the large targeting vector comprises a human DNA sequence that is homologous to a non-human DNA sequence of a non-human eukaryotic cell;

(b) introducing the large targeting vector into the non-human eukaryotic cell to modify an endogenous chromosomal locus of interest in the cell to form a modified allele comprising the human DNA sequence; and

,

(c) assaying the cell for the modified allele.

2. The method of claim 1, wherein the non-human eukaryotic cell is a mammalian embryonic stem cell.

3. The method of claim 2, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell.

4. The method of claim 3, wherein the rodent embryonic stem cell is selected from a mouse embryonic stem cell and a rat embryonic stem cell.

5. The method of claim 1, wherein the large cloned genomic fragment comprises a human gene or gene fragment.

6. The method of claim 1, wherein the DNA sequence that is homologous to a DNA sequence of the non-human eukaryotic cell is an orthologous coding sequence.

7. The method of claim 6, wherein the method is employed to replace a murine genetic locus with an orthologous human genetic locus.

8. The method of claim 1, wherein the method is employed to insert a new coding region, insert or replace a gene segment, or insert or replace a regulatory element.

9. The method of claim 1, wherein the method is employed to replace an endogenous locus.

10. The method of claim 1, wherein the method is employed to create a conditional allele.

11. The method of claim 1, wherein the large targeting vector is capable of accommodating DNA fragments greater than 20 kb.

12. The method of claim 1, wherein the large targeting vector is capable of accommodating DNA fragments greater than 100 kb.

13. The method of claim 1, wherein the large targeting vector is greater than 140 kb.

14. The method of claim 1, wherein the large targeting vector is greater than 190 kb.

15. The method of claim 1, wherein the large targeting vector is greater than 235 kb.

16. The method of claim 1, wherein the large targeting vector is greater than 256 kb.

17. The method of claim 1, wherein the large targeting vector is greater than 265 kb.

18. The method of claim 1, wherein the large targeting vector is greater than 288 kb.

19. The method of claim 1, wherein the large targeting vector is greater than 308 kb.

20. The method of claim 1, wherein the large targeting vector is greater than 322 kb.

21. The method of claim 1, wherein the large targeting vector is greater than 368 kb.

22. The method of claim 1, wherein assaying the cell for the modified allele comprises detecting modification of a parental allele.

23. The method of claim 1, wherein the large targeting vector comprises a sequence selected from a loxP site and a FRT site.

24. A method for targeting a large cloned genomic fragment to a desired position in a non-human genome, comprising

(a) using bacterial homologous recombination to precisely engineer a genetic modification within a large cloned genomic fragment to form a large targeting vector, wherein the large targeting vector comprises a human DNA sequence that is orthologous to a non-human DNA sequence of a non-human eukaryotic cell;

(b) introducing the large targeting vector into the non-human eukaryotic cell to modify an endogenous chromosomal locus of interest in the cell to form a modified allele comprising the human DNA sequence; and

,

(c) assaying the cell for the modified allele.

25. The method of claim 24, wherein the non-human eukaryotic cell is a mammalian embryonic stem cell.

26. The method of claim 24, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell.

27. The method of claim 26, wherein the rodent embryonic stem cell is selected from a mouse embryonic stem cell and a rat embryonic stem cell.

28. The method of claim 24, wherein the large cloned genomic fragment comprises a human gene or gene fragment or a mouse gene or gene fragment.

29. The method of claim 24, wherein the orthologous human DNA sequence is a gene coding sequence.

30. The method of claim 27, wherein the method is employed to replace a murine genetic locus with an orthologous human genetic locus.

31. The method of claim 24, wherein the method is employed to insert a new coding region, insert or replace a gene segment, or insert or replace a regulatory element.

32. The method of claim 24, wherein the method is employed to replace an endogenous locus.

33. The method of claim 24, wherein the method is employed to create a conditional allele.

34. The method of claim 24, wherein the large targeting vector is capable of accommodating DNA fragments greater than 20 kb.

35. The method of claim 24, wherein the large targeting vector is capable of accommodating DNA fragments greater than 100 kb.

36. The method of claim 24, wherein the large targeting vector is greater than 140 kb.

37. The method of claim 24, wherein the large targeting vector is greater than 190 kb.

38. The method of claim 24, wherein the large targeting vector is greater than 235 kb.

39. The method of claim 24, wherein the large targeting vector is greater than 256 kb.

40. The method of claim 24, wherein the large targeting vector is greater than 265 kb.

41. The method of claim 24, wherein the large targeting vector is greater than 288 kb.

42. The method of claim 24, wherein the large targeting vector is greater than 308 kb.

43. The method of claim 24, wherein the large targeting vector is greater than 322 kb.

44. The method of claim 24, wherein the large targeting vector is greater than 368 kb.

45. The method of claim 24, wherein assaying the cell for the modified allele comprises detecting modification of a parental allele.

46. The method of claim 24, wherein the large targeting vector comprises a sequence selected from a loxP site and a FRT site.

47. A method for modifying a desired position in a non-human genome, comprising

(a) using bacterial homologous recombination to precisely engineer a genetic modification within a large cloned genomic fragment to form a large targeting vector,

(b) introducing the large targeting vector into the non-human eukaryotic cell to modify an endogenous chromosomal locus of interest in the cell to form a modified allele comprising the large targeting vector;

and,

(c) assaying the cell for the modified allele.

48. The method of claim 47, wherein the modification is a deletion of a coding sequence, gene segment or regulatory element.

49. The method of claim 47, wherein the modification is an alteration of a coding sequence, gene segment or regulatory element.

50. The method of claim 49, wherein the alteration comprises substitution of an endogenous gene or locus with an orthologous gene or locus.

51. The method of claim 47, wherein the modification comprises an insertion of a coding region, gene segment or regulatory element.

52. The method of claim 51, wherein the modification comprises an insertion of a reporter gene into an endogenous gene, wherein the reporter gene is under transcriptional control of the endogenous gene.

53. The method of claim 52, wherein the reporter gene is β

-galactosidase (LacZ).

54. The method of claim 47, wherein the modification comprises the creation of a conditional allele.

55. The method of claim 47, wherein the modification comprises the introduction of loxP and/or FRT sites.

56. The method of claim 47, wherein the modification comprises the replacement of an endogenous coding sequence or gene segment with an orthologous coding sequence or gene segment.