Methods of modifying eukaryotic cells

• US 20050144655A1
• Filed: 10/31/2001
• Published: 06/30/2005
• Est. Priority Date: 10/31/2000
• Status: Abandoned Application

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Yancopoulos, George D., Murphy, Andrew J., Economides, Aris N., Frendewey, David, Valenzuela, David M.
• Application Number:
  US10/415,440
• Publication Number:
  US 20050144655A1
• Time in Patent Office: Days
• US Class Current: 800/8
• 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 genes(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 gene or chromosomal locus of interest in eukaryotic cells, comprising:

a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;

b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the eukaryotic cells (LTVEC);

c) introducing the LTVEC of (b) into the eukaryotic cells to modify the endogenous gene or chromosomal locus in the cells; and

d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified.

2. The method of claim 1 wherein the large cloned genomic fragment containing a DNA sequence is homologous to the endogenous gene or chromosomal locus of interest.

3. The method of claim 1 wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element;

alteration of a coding sequence, gene segment, or regulatory element;

insertion of a new coding sequence, gene segment, or regulatory element;

creation of a conditional allele;

or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.

4. The method of claim 3 wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.

5. The method of claim 4 wherein the fusion comprises an epitope tag or bifunctional protein.

6. The method of claim 1 wherein the quantitative assay comprises quantitative PCR, FISH, comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe, Invader Probes®

, or MMP assays®

.

7. The method of claim 6 wherein the quantitative PCR comprises TaqMan®

, Molecular Beacon, or Eclipse™

probe technology.

8. The method of claim 1 wherein the eukaryotic cell is a mammalian embryonic stem cell.

9. The method of claim 8 wherein the embryonic stem cell is a mouse, rat, or other rodent embryonic stem cell.

10. The method of claim 1 wherein the endogenous gene or chromosomal locus is a mammalian gene or chromosomal locus.

11. The method of claim 10 wherein the endogenous gene or chromosomal locus is a human gene or chromosomal locus.

12. The method of claim 10 wherein the endogenous gene or chromosomal locus is a mouse, rat, or other rodent gene or chromosomal locus.

13. The method of claim 1 wherein the LTVEC is capable of accommodating large DNA fragments greater than 20 kb.

14. The method of claim 13 wherein the LTVEC is capable of accommodating large DNA fragments greater than 100 kb.

16. A genetically modified endogenous gene or chromosomal locus produced by the method of any one of claims 1 or 15.

17. The genetically modified endogenous gene or chromosomal locus of claim 16 wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element;

alteration of a coding sequence, gene segment, or regulatory element;

insertion of a new coding sequence, gene segment, or regulatory element;

creation of a conditional allele;

or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.

18. The genetically modified endogenous gene or chromosomal locus of claim 17 wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.

19. The genetically modified endogenous gene or chromosomal locus of claim 18 wherein the fusion comprises an epitope tag or bifunctional protein.

20. A genetically modified eukaryotic cell produced by the method of claim 1.

22. The genetically modified eukaryotic cell of claim 20 wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element;

alteration of a coding sequence, gene segment, or regulatory element;

insertion of a new coding sequence, gene segment, or regulatory element;

creation of a conditional allele;

or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.

23. The genetically modified eukaryotic cell of claim 22 wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.

24. The genetically modified eukaryotic cell of claim 23 wherein the fusion comprises an epitope tag or bifunctional protein.

25. A non-human organism containing a genetically modified endogenous gene or chromosomal locus produced by the method of claim 1.

27. A non-human organism produced from the genetically modified eukaryotic cell of any one of claims 20, 22, 23, or 24.

29. A genetically modified embryonic stem cell produced by the method of claim 1.

30. The genetically modified embryonic stem cell of claim 29 wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element;

alteration of a coding sequence, gene segment, or regulatory element;

insertion of a new coding sequence, gene segment, or regulatory element;

creation of a conditional allele;

or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from a different species.

31. The genetically modified embryonic stem cell of claim 30 wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.

32. The genetically modified embryonic stem cell of claim 31 wherein the fusion comprises an epitope tag or bifunctional protein.

38. The non-human organism of any one of claims 25, 33, 35, or 36 wherein the non-human organism is a mouse, rat, or other rodent.

49. The use of the genetically modified eukaryotic cell of claim 20 for the production of a non-human organism.

51. The use of the genetically modified embryonic stem cell of claim 29 for the production of a non-human organism.

52. The method of claims 1 or 15, wherein about 1-5 μ

g of the large targeting vector of (c) is introduced to about 1×

10⁷ cells.

15. A method for genetically modifying an endogenous gene or chromosomal locus of interest in mouse embryonic stem cells, comprising:

a) obtaining a large cloned genomic fragment greater than 20 kb which contains a DNA sequence of interest, wherein the large cloned DNA fragment is homologous to the endogenous gene or chromosomal locus;

b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the mouse embryonic stem cells, wherein the genetic modification is deletion of a coding sequence, gene segment, or regulatory element;

c) introducing the large targeting vector of (b) into the mouse embryonic stem cells to modify the endogenous gene or chromosomal locus in the cells; and

d) using a quantitative assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (c) to identify those mouse embryonic stem cells in which the endogenous gene or chromosomal locus has been genetically modified, wherein the quantitative assay is quantitative PCR.

21. A genetically modified mouse embryonic stem cell produced by the method of claim 15.

26. A mouse containing a genetically modified endogenous gene or chromosomal locus produced by the method of claim 15.

28. A mouse produced from the genetically modified mouse embryonic stem cell of claim 21.

50. The use of the genetically modified mouse embryonic stem cell of claim 21 for the production of a mouse.

33. A non-human organism containing a genetically modified endogenous gene or chromosomal locus of interest, produced by a method comprising the steps of:

a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;

b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector (LTVEC) for use in embryonic stem cells;

c) introducing the LTVEC of (b) into the embryonic stem cells to modify the endogenous gene or chromosomal locus in the cells;

d) using a quantitative assay to detect modification of allele (MOA) in the embryonic stem cells of (c) to identify those embryonic stem cells in which the endogenous gene or chromosomal locus has been genetically modified;

e) introducing the embryonic stem cell of (d) into a blastocyst; and

f) introducing the blastocyst of (e) into a surrogate mother for gestation.

37. The non-human organism of any one of claims 33, 35, or 36 wherein the large cloned genomic fragment containing a DNA sequence is homologous to the endogenous gene or chromosomal locus of interest.

39. The non-human organism of claim 33 wherein the blastocyst is a mouse, rat, or other rodent blastocyst.

41. The non-human organism of any one of claims 33, 35, or 36 wherein the surrogate mother is a mouse, rat, or other rodent.

42. The non-human organism of claim 33 wherein the embryonic stem cell is a mammalian embryonic stem cell.

43. The non-human organism of claim 42 wherein the mammalian embryonic stem cell is a mouse, rat, or other rodent embryonic stem cell.

44. The non-human organism of any one of claims 33, 35, or 36 wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element;

alteration of a coding sequence, gene segment, or regulatory element;

insertion of a new coding sequence, gene segment, or regulatory element;

creation of a conditional allele;

or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.

45. The non-human-organism of claim 44 wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.

46. The non-human organism of claim 45 wherein the fusion comprises an epitope tag or bifunctional protein.

47. The non-human organism of any one of claims 33, 35, or 36 wherein the quantitative assay comprises quantitative PCR, FISH, comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe, Invader Probes®

, or MMP®

assays.

48. The non-human organism of claim 47 wherein the quantitative PCR comprises TaqMan®

, Molecular Beacon, or Eclipse™

probe technology.s.

54. The non-human organism of claims 33, 35, or 36, wherein about 1-5 μ

g of the large targeting vector of (c) is introduced to about 1×

10⁷ cells.

34. A mouse containing a genetically modified endogenous gene or chromosomal locus of interest, produced by a method comprising the steps of:

a) obtaining a large cloned genomic fragment greater than 20 kb which contains a DNA sequence of interest, wherein the large cloned DNA fragment is homologous to the endogenous gene or chromosomal locus;

b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the mouse embryonic stem cells, wherein the genetic modification is deletion of a coding sequence, gene segment, or regulatory element;

c) introducing the large targeting vector of (b) into the mouse embryonic stem cells to modify the endogenous gene or chromosomal locus in the cells; and

d) using a quantitative assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (c) to identify those mouse embryonic stem cells in which the endogenous gene or chromosomal locus has been genetically modified, wherein the quantitative assay is quantitative PCR;

e) introducing the mouse embryonic stem cell of (d) into a blastocyst; and

f) introducing the blastocyst of (e) into a surrogate mother for gestation.

53. The mouse of claim 34, wherein about 1-5 μ

g of the large targeting vector of (c) is introduced to about 1×

10⁷ cells.

35. A non-human organism containing a genetically modified endogenous gene or chromosomal locus, produced by a method comprising the steps of:

a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;

b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC);

c) introducing the LTVEC of (b) into the eukaryotic cells to genetically modify the endogenous gene or chromosomal locus in the cells;

d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified;

e) removing the nucleus from the eukaryotic cell of (d);

f) introducing the nucleus of (e) into an oocyte; and

g) introducing the oocyte of (f) into a surrogate mother for gestation.

40. The non-human organism of claim 35 wherein the oocyte is a mouse, rat, or other rodent oocyte.

36. A non-human organism containing a genetically modified endogenous gene or chromosomal locus, produced by a method comprising the steps of:

a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;

b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC);

c) introducing the LTVEC of (b) into the eukaryotic cells to genetically modify the endogenous gene or chromosomal locus in the cells;

d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified;

e) fusing the eukaryotic cell of (d) with another eukaryotic cell; and

f) introducing the fused eukaryotic cell of (e) into a surrogate mother for gestation.