Chimeric mice that are produced by microcell mediated chromosome transfer and that retain a human antibody gene

• US 6,632,976 B1
• Filed: 03/02/1998
• Issued: 10/14/2003
• Est. Priority Date: 08/29/1995
• Status: Expired due to Term

• Current Assignee: Kyowa Hakko Kirin Company Limited (Kirin Holdings Company Limited)
• Original Assignee: Kirin Beer Kabushiki Kaisha
• Inventors: Tomizuka, Kazuma, Yoshida, Hitoshi, Ishida, Isao, Hanaoka, Kazunori, Oshimura, Mitsuo
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US09/033,936
• Time in Patent Office: 2,052 Days
• Field of Search: 800/21, 800/22, 800/25, 800/13, 800/4, 800/5, 800/6, 800/18, 435/455, 435/463, 435/70.1, 435/70.2, 435/70.21
• US Class Current: 800/18
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• 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 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2517/02 Cells from transgenic animals
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

A chimeric, non-human animal can be produced by a method that entails providing a microcell that contains one or more foreign chromosomes or fragment(s) thereof and then fusing the microcell with a pluripotent cell, thereby introducing the foreign chromosome(s) or fragment(s) into the latter. The pluripotent cell thus obtained can be used to generate a chimeric, non-human animal, the cells, tissues, and/or progeny of which can be the source of a product, such as an antibody, that is associated with one or more genes on the foreign chromosome(s) or fragment(s).

Claims

1. A method for producing a chimeric mouse wherein at least some mouse cells retain a foreign chromosome(s) or a fragment(s) thereof, which method comprises:

1) preparing a microcell retaining a foreign chromosome(s) or a fragments thereof that comprise a human antibody gene;

2) transferring the foreign chromosome(s) or fragment(s) thereof into a pluripotent mouse cell by fusion with the microcell;

3) transferring the pluripotent mouse cell into a mouse embryo;

4) transferring the mouse embryo into a uterus or oviduct of a pseudo-pregnant female mouse; and

5) allowing said mouse embryo to develop into said chimeric mouse.

3. The method of claim 1 or 2, wherein the foreign chromosome(s) or fragment(s) thereof is human-derived and has a base length not less than 1 Mb.

4. The method of claim 1 or 2, wherein the microcell retaining a foreign chromosome(s) or a fragment(s) thereof is induced from a hybrid cell prepared by the fusion of a cell from which the foreign chromosome(s) or fragment(s) thereof is derived, with a cell having a high ability to form a microcell.

5. The method of claim 4, wherein the microcell retaining a foreign chromosome(s) or a fragment(s) thereof is induced from a cell prepared by a further fusion of the microcell induced from the hybrid cell with a cell having a high ability to form a microcell.

6. The method of claim 4, wherein the cell having a high ability to form a microcell is a mouse A9 cell.

7. The method of claim 1 or 2, wherein the foreign chromosome(s) or fragment(s) thereof is derived from a human normal diploid cell.

8. The method of claim 1 or 2, wherein the pluripotent mouse cell is an ES cell.

9. The method of claim 1, wherein the pluripotent mouse cell has a disrupted endogenous antibody gene that is homologous to the human antibody gene.

12. The method of claim 9, wherein the mouse embryo for the production of the chimeric mouse is derived from a mouse strain deficient in an endogenous antibody gene that is homologous to the human antibody gene.

13. The method of claim 12, wherein the mouse strain deficient in the endogenous antibody gene is produced by homologous recombination.

14. The method of claim 1, wherein the chimeric mouse expresses the human antibody gene.

15. The method of claim 1, wherein the chimeric mouse can transmit the foreign chromosome(s) or fragment(s) thereof to its progeny.

16. The method of claim 1, wherein the foreign chromosome(s) or a fragment(s) is independently maintained from the mouse chromosomes.

17. The method of claim 1, wherein the foreign chromosome(s) or a fragment(s) has a base length not less than 1 Mb.

18. The method of claim 1, wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ

(kappa) gene, human light-chain λ

(lambda) gene and combinations thereof.

2. A method for producing a pluripotent mouse cell retaining a foreign chromosome(s) or a fragment(s) thereof, which comprises preparing a microcell retaining a foreign chromosome(s) or a fragment(s) thereof and transferring the foreign chromosome(s) or fragment(s) thereof into a pluripotent mouse cell by fusion with the microcell, wherein the foreign chromosome(s) or fragment(s) thereof comprise a human antibody gene.

10. The method of claim 2, wherein the pluripotent mouse cell has a disrupted endogenous antibody gene that is homologous to the human antibody gene.

11. The method of claim 9 or 10, wherein the endogenous antibody gene is disrupted by homologous recombination.

19. The method of claim 2, wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ

(kappa) gene, human light-chain λ

(lambda) gene and combinations thereof.

20. A pluripotent mouse cell which retains a human-derived chromosome(s) or a fragment(s) thereof having a base length not less than 1 Mb, and which can express the gene(s) on the human-derived chromosome(s) or fragment(s) thereof after the differentiation of the cell, wherein the human-derived chromosome(s) or fragment(s) thereof comprise a human antibody gene.

21. The pluripotent mouse cell of claim 20, wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ

(kappa) gene, human light-chain λ

(lambda) gene and combinations thereof.

23. The mouse of claim 21, wherein the foreign chromosome(s) or a fragment(s) has a base length not less than 1 Mb.

24. The mouse of claim 21, wherein the foreign chromosome(s) or a fragment(s) is independently maintained from the mouse chromosomes.

22. A mouse wherein at least some mouse cells retain a foreign chromosome(s) or a fragment(s) thereof, wherein said foreign chromosome(s) or a fragment(s) thereof is independently maintained from the mouse chromosomes and/or has a base length not less than 1 Mb, and wherein the foreign chromosome(s) or fragment(s) thereof comprise a human antibody gene.

25. The mouse of claim 22, wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ

(kappa) gene, human light-chain λ

(lambda) gene and combinations thereof.

26. The mouse of claim 22, wherein the foreign chromosome is a human-derived chromosome.

29. A mouse which can be produced by mating the mice of any one of claims 22, 26, or 27, wherein at least some of the cells of the mouse retain a foreign or human-derived chromosome(s) or a fragment(s) thereof, which has a base length not less than 1 Mb and/or is independently maintained from the mouse chromosomes, and wherein the foreign chromosome(s) or fragment(s) thereof comprise a human antibody gene.

30. The mouse of claim 29, wherein the foreign chromosome(s) or a fragment(s) has a base length not less than 1 Mb.

31. The mouse of claim 29, wherein the foreign chromosome(s) or a fragment(s) is independently maintained from the mouse chromosomes.

32. A progeny of the mouse of claim 29, wherein at least some of the cells of the progeny retain a foreign or human-derived chromosome(s) or a fragment(s) thereof, which has a base length not less than 1 Mb and/or is independently maintained from the mouse chromosomes.

33. The progeny of claim 32, wherein the foreign chromosome(s) or a fragment(s) has a base length not less than 1 Mb.

34. The progeny of claim 32, wherein the foreign chromosome(s) or a fragment(s) is independently maintained from the mouse chromosomes.

35. A progeny mouse which can be produced by mating the mouse of any one of claims 22, 26, or 27 with a mouse strain deficient in an endogenous antibody gene that is homologous to the human antibody gene, wherein at least some cells of the progeny mouse retain the foreign or human-derived chromosome(s) or fragment(s) thereof, which has a base length not less than 1 Mb and/or is independently maintained from the mouse chromosomes.

36. A progeny of the mouse of claim 35, wherein the progeny retains a foreign or human-derived chromosome(s) or a fragment(s) thereof, which has a base length not less than 1 Mb and/or is independently maintained from the mouse chromosomes, and wherein the progeny is deficient in the endogenous antibody gene.

37. The progeny mouse of claim 35, wherein the foreign chromosome(s) or a fragment(s) has a base length not less than 1 Mb.

38. The progeny mouse of claim 35, wherein the foreign chromosome(s) or a fragment(s) is independently maintained from the mouse chromosomes.

41. A method for producing an antibody, which comprises expressing a foreign or human-derived gene in the mouse of any one of claims 22, 26, 27 and 39, the tissue thereof, the cell thereof, the B cell thereof or the hybridoma prepared by the fusion of the B cell thereof with a myeloma cell, and collecting the antibody as an expression product.

42. A method for producing an antibody, which comprises expressing a foreign or human-derived gene in the progeny mouse of claim 35, the tissue thereof, the cell thereof, the B cell thereof or the hybridoma prepared by the fusion of the B cell thereof with a myeloma cell, and collecting the antibody as an expression product.

43. A method for producing an antibody, which comprises expressing a foreign or human-derived gene in the progeny of claim 36, the tissue thereof, the cell thereof, the B cell thereof or the hybridoma prepared by the fusion of the B cell thereof with a myeloma cell, and collecting the antibody as an expression product.

27. A mouse wherein at least some mouse cells retain a human-derived chromosome(s) or a fragment(s) thereof having a base length not less than 1 Mb, and which can express the gene(s) on the human-derived chromosome(s) or fragment(s) thereof, wherein at least one of the genes expressed on the human-derived chromosome(s) or fragment(s) is a human antibody gene.

28. The mouse of claim 27, wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ

(kappa) gene, human light-chain λ

(lambda) gene and combinations thereof.

39. A mouse wherein at least some mouse cells retain a human-derived DNA fragment(s) comprising human antibody gene, having a base length not less than 1 Mb and/or independently maintained from the mouse chromosome, wherein the mouse is capable of expressing the human antibody gene.

40. The mouse of claim 39, wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ

(kappa) gene, human light-chain λ

(lambda) gene and combinations thereof.

44. A mouse wherein at least some mouse cells retain a human-derived chromosome(s) or a fragment(s) thereof containing a human antibody gene and having a base length not less than 1 Mb, and which can express at least one class or subclass of the human antibody.

45. The mouse of claim 44, wherein the foreign chromosome(s) or a fragment(s) is independently maintained from the mouse chromosomes.

47. The mouse of claim 44 or 46, wherein the human antibody gene is selected from the group consisting of human heavy-chain gene, human light-chain κ

(kappa) gene, human light-chain λ

(lambda) gene and combinations thereof.

48. The mouse of claim 44 or 46, wherein at least some mouse cells retain the entire region of the unrearranged human antibody heavy-chain gene locus.

49. The mouse of claim 44 or 46, wherein at least some mouse cells retain the entire region of the unrearranged human antibody light-chain κ

(kappa) gene locus.

50. The mouse of claim 44 or 46, wherein at least some mouse cells retain the entire region of the unrearranged human antibody light-chain λ

(lambda) gene locus.

51. The mouse of claim 44 or 46, in the genome of which there is a disrupted, endogenous antibody gene.

52. The mouse of claim 51, wherein the disruption of the endogenous antibody gene is by homologous recombination.

53. A method for preparing a human polyclonal antibody, which comprises immunizing the mouse of claim 44 or 46 with an antigen, and collecting anti-serum from the blood of the mouse.

54. A method for preparing anti-serum containing a human antibody, which comprises immunizing the mouse of claims 44 or 46 with an antigen, and collecting an anti-serum from the blood of the mouse.

46. A mouse wherein at least some mouse cells retain a human antibody gene having a base length not less than 1 Mb, and which can express at least one class or subclass of the human antibody.