Methods for producing transgenic animals

• US 20030221206A1
• Filed: 04/21/2003
• Published: 11/27/2003
• Est. Priority Date: 12/17/1999
• Status: Abandoned Application

• Current Assignee: Emory University, Magee-Womens Health Corporation
• Original Assignee: Oregon Health & Science University
• Inventors: Chan, Anthony W.S., Schatten, Gerald
• Application Number:
  US10/421,357
• Publication Number:
  US 20030221206A1
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 9/02 Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos

Abstract

The present invention relates to methods for producing transgenic animals. Specifically, the methods of the present invention include production of a transgenic animal by transgenic intracytoplasmic sperm injection, retroviral gene transfer, intracytoplasmic nuclear injection, and pronuclear injection. In addition, the present invention also relates to methods for using transgenic animals as models for human disease and diagnosis. In particular, these transgenic animals may be used as models for embryo and fetal development, as models to assess the safety and efficacy of drug therapy and gene therapy, and as models for disease diagnosis. The methods of the present invention are also directed to methods of using transgenic embryonic cells to treat human diseases.

Claims

1. A method for producing a transgenic animal comprising the steps of:

transferring transgenic embryos, which were produced by transferring exogenous DNA from spermatozoa, to oocytes by intracytoplasmic sperm injection and cultured to an embryonic stage, to the oviducts of surrogate females; and

producing a transgenic animal by parturition.

2. The method of claim 1, wherein said animal is selected from the group consisting of mammals, birds, reptiles, amphibians, and fish.

3. The method of claim 2, wherein said animal is a nonhuman primate.

4. The method of claim 3, wherein said nonhuman primate is selected from the group consisting of rhesus macaque, baboon, capuchin, chimpanzee, pigtail macaque, sooty mangabey, squirrel monkey, orangutan.

5. The method of claim 1, wherein said exogenous DNA is an expression vector.

6. The method of claim 5, wherein said expression vector comprises regulatory nucleic acid sequences and a structural gene sequence.

7. The method of claim 5, wherein said expression vector is selected from the group consisting of plasmid vectors, viral vectors, and retroviral vectors.

8. The method of claim 6, wherein said regulatory nucleic acid sequence is a promoter.

9. The method of claim 8, wherein said promoter is a viral promoter.

10. The method of claim 9, wherein said viral promoter is the cytomegalovirus promoter.

11. The method of claim 8, wherein said promoter is the protamine-1 promoter.

12. The method of claim 8, wherein said promoter is selected from the group consisting of inducible promoter and constitutive promoter.

13. The method of claim 6, wherein said structural gene sequence encodes a polypeptide selected from the group consisting of receptors, enzymes, cytokines, hormones, growth factors, immunoglobulins, cell cycle proteins, cell signaling proteins, membrane proteins, and cytoskeletal proteins.

14. The method of claim 6, wherein said structural gene sequence is a reporter gene.

15. The method of claim 14, wherein said reporter gene is green fluorescent protein gene.

16. The method of claim 14, wherein said reporter gene is selected from the group consisting of β

-galactosidase gene, secreted placental alkaline phosphatase gene, and luciferase gene.

17. The method of claim 14, wherein said reporter gene is used to monitor the development of a cell or tissue in a transgenic embryo.

18. The method of claim 6, wherein said structural gene sequence is a disease gene.

19. The method of claim 18, wherein said disease gene is linked to a disease selected from the group consisting of cardiovascular diseases, neurological diseases, reproductive disorders, cancers, eye diseases, endocrine disorders, pulmonary diseases, metabolic disorders, hereditary diseases, autoimmune disorders, and aging.

20. The method of claim 1, wherein said exogenous DNA is labeled with a fluorophore.

21. The method of claim 20, wherein said fluorophore is rhodamine.

22. The method of claim 1, wherein said exogenous DNA consists of one or more expression vectors.

23. The method of claim 5, wherein said expression vector comprises regulatory nucleic acid sequences and two or more structural gene sequences.

24. The method of claim 1, wherein said oocyte is cultured to the 3-16 cell embryo stage.

25. The method of claim 1, wherein said transgenic animal is a model for human disease.

26. The method of claim 25, wherein said human disease is selected from the group consisting of cardiovascular diseases, neurological diseases, reproductive disorders, cancers, eye diseases, endocrine disorders, pulmonary diseases, metabolic disorders, autoimmune disorders, and aging.

27. The method of claim 1, wherein said transgenic animal is a model for hereditary disease.

28. The method of claim 1, wherein said transgenic animal is a model for embryo and fetal development.

29. The method of claim 1, wherein said transgenic animal is model to demonstrate the safety and efficacy of treatments selected from the group comprising drug therapy, gene therapy, stem cell therapy, and somatic cell therapy.

30. The method of claim 1, wherein said transgenic animal is model for disease diagnosis.

31. The method of claim 1, wherein said method is used to preserve an endangered species.

32. The method of claim 1, wherein said method is used for sperm-mediated gene therapy.

33. The method of claim 1, wherein the said spermatazoa are subjected to sanitizing treatments selected from the group consisting of chemical decontamination and physical removal.

34. The method of claim 33, wherein said chemical decontamination is selected from the group consisting of proteinases, DNases, and RNases.

35. The method of claim 33, wherein said sanitizing treatment by physical removal is selected from the group consisting of polystyrene and magnetic beads.

36. A transgenic embryo produced according to the method of claim 1.

37. The transgenic embryo of claim 36, wherein said transgenic embryo is a model for embryo and fetal development.

38. A transgenic animal produced according to the method of claim 1.

39. The transgenic animal of claim 38, wherein said transgenic animal is a model for human disease.

40. The transgenic animal of claim 38, wherein said transgenic animal is a model for hereditary disease.

41. The transgenic animal of claim 38, wherein said transgenic animal is model to demonstrate the safety and efficacy of treatments selected from the group consisting of drug therapy, gene therapy, stem cell therapy, and somatic cell therapy.

42. The transgenic animal of claim 38, wherein said transgenic animal is model for disease diagnosis.

43. The transgenic embryo of claim 36, wherein said transgenic embryo is a transgenic chimeric embryo.

79. The method of claim 1, wherein said exogenous DNA is bound to spermatozoa comprising the steps of:

mixing exogenous DNA with spermatozoa;

incubating DNA-spermatozoa mixture for 30 minutes at 37°

C.; and

washing DNA-bound spermatozoa in TALP-HEPES buffer.

44. A method for producing a transgenic animal comprising the steps of:

transferring transgenic embryos, which were produced by transferring exogenous DNA to oocytes by injection of a retroviral vector, fertilizing the oocytes by intracytoplasmic sperm injection, and culturing said oocytes to the embryonic stage, to the oviducts of surrogate females; and

producing a transgenic animal by parturition.

45. The method of claim 44, wherein said animal is a nonhuman primate.

46. The method of claim 45, wherein nonhuman primate is selected from the group consisting of rhesus macaque, baboon, capuchin, chimpanzee, pigtail macaque, sooty mangabey, squirrel monkey, orangutan.

47. The method of claim 44, wherein said oocyte comprises a prematuration oocyte.

48. The method of claim 44, wherein said oocyte comprises a prefertilization oocyte.

49. The method of claim 44, wherein the perivitelline space of said oocyte is injected with a retroviral vector.

50. The method of claim 44, wherein said oocyte is cultured to the 4-8 cell embryo stage.

51. The method of claim 44, wherein said retroviral vector comprises regulatory gene sequences and structural gene sequences.

52. The method of claim 51, wherein said regulatory sequence is a promoter.

53. The method of claim 52, wherein said promoter is a viral promoter.

54. The method of claim 53, wherein said viral promoter is the cytomegalovirus promoter.

55. The method of claim 52, wherein said promoter is a human elongation factor-1 alpha promoter.

56. The method of claim 44, wherein said retroviral vector is selected from the group consisting of Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus.

57. The method of claim 44, wherein detection of said retroviral vector is determined by a retroviral assay selected from the group consisting of CV-1/S+L-assay, PCR, Southern analysis, and clonal CV-1-LNC-EGFP cells.

58. The method of claim 44, wherein said retroviral vector comprises a membrane-associated protein.

59. The method of claim 58, wherein said membrane-associated protein is a glycoprotein selected from Rhabdoviridae.

60. The method of claim 59, wherein said glycoprotein is obtained from the group consisting of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus, Rabies virus, and Mokola virus.

61. The method of claim 51, wherein said structural gene sequence is a reporter gene.

62. The method of claim 61, wherein said reporter gene is green fluorescent protein gene.

63. The method of claim 61, wherein said reporter gene is selected from the group consisting of β

-galactosidase gene, secreted placental alkaline phosphatase gene, and luciferase gene.

64. The method of claim 51, said structural gene sequence encodes a polypeptide selected from the group comprising receptors, enzymes, cytokines, hormones, growth factors, immunoglobulins, cell cycle proteins, cell signaling proteins, membrane proteins, and cytoskeletal proteins.

65. A method for producing a transgenic primate comprising the steps of:

transferring said transgenic embryos which were produced by dissociating blastomeres from an embryo, removing the nuclei from the blastomeres, injecting the blastomere nuclei into enucleated oocytes by intracytoplasmic nuclear injection, and activating and culturing oocytes to an embryonic stage, to the oviducts of surrogate females; and

producing a transgenic primate by parturition.

66. The method of claim 65, wherein said primate is a nonhuman primate.

67. The method of claim 66, wherein nonhuman primate is selected from the group consisting of rhesus macaque, baboon, capuchin, chimpanzee, pigtail macaque, sooty mangabey, squirrel monkey, orangutan, and members thereof.

68. The method of claim 65, wherein inner cell mass cells are isolated from said blastomere for nuclear transfer.

69. The method of claim 65, wherein said oocyte activation is selected from the group consisting of chemical activation, sperm cytosolic (oscillin) activation, and electrical activation.

70. A method for producing a transgenic primate comprising the steps of:

transferring transgenic embryos, which were produced by isolating nuclei from somatic cells, injecting the nuclei into enucleated oocytes by intracytoplasmic nuclear injection, then activating and culturing the oocytes to an embryo stage, to the oviduct of surrogate females; and

producing a transgenic primate by parturition.

71. The method of claim 70, wherein said primate is a nonhuman primate.

72. The method of claim 71, wherein nonhuman primate is selected from the group consisting of rhesus monkey, baboon, capuchin, chimpanzee, pigtail macaque, sooty mangabey, squirrel monkey, orangutan.

73. The method of claim 70, wherein said somatic cells are skin cells.

74. The method of claim 74, wherein said oocyte activation is selected from the group comprising chemical activation, sperm cytosolic (oscillin) activation, and electrical activation.

75. A method for producing a transgenic primate comprising the steps of:

transferring transgenic embryos, which were produced by fertilizing an oocyte by intracytoplasmic sperm injection, then transferring exogenous DNA to the pronucleus of fertilized oocyte by pronuclear injection, then culturing the fertilized oocyte to an embryonic stage, to oviduct of surrogate females; and

producing a transgenic primate by parturition.

76. A method of producing transgenic primate cells wherein said transgenic cells are used to treat human diseases.

77. The method of claim 76, wherein said transgenic primate cells are produced by methods selected from the group consisting of intracytoplasmic sperm injection, retroviral gene transfer, intracytoplasmic nuclear injection, and pronuclear injection.

78. The method of claim 76, wherein said human disease is selected from the group consisting of cardiovascular disease, neurological diseases, reproductive disorders, cancer, eye diseases, endocrine disorders, pulmonary disease, metabolic disorders, autoimmune disorders, and aging.