Porcine CMP-N-Acetylneuraminic acid hydroxylase gene

• US 20050223418A1
• Filed: 06/07/2004
• Published: 10/06/2005
• Est. Priority Date: 06/06/2003
• Status: Active Grant

• Current Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Original Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Inventors: Koike, Chihiro
• Granted Patent: US 7,368,284 B2
• Time in Patent Office: Days
• US Class Current: 800/8
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/108 Swine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 2267/025 Animal producing cells or organs for transplantation
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)

Abstract

The present invention provides porcine CMP-N-Acetylneuraminic-Acid Hydroxylase (CMP-Neu5Ac hydroxylase) protein, cDNA, and genomic DNA regulatory sequences. Furthermore, the present invention includes porcine animals, tissues, and organs, as well as cells and cell lines derived from such animals, tissues, and organs, which lack expression of functional CMP-Neu5Ac hydroxylase. Such animals, tissues, organs, and cells can be used in research and in medical therapy, including in xenotransplantation, and in industrial livestock farming operations.

Claims

1. An isolated full length cDNA sequence encoding a porcine CMP-N-Acetyneuraminic-Acid hydroxylase protein.

2. The cDNA of claim 1 wherein the sequence is SEQ ID NO 1.

3. The cDNA of claim 1 wherein the sequence is SEQ ID NO 3.

4. The cDNA of claim 1 wherein the sequence is SEQ ID NO 5.

5. The cDNA of claim 1 wherein the sequence is SEQ ID NO 7.

6. An isolated amino acid sequence encoding a CMP-N-Acetyneuraminic-Acid hydroxylase protein wherein the sequence is SEQ ID NO 2.

7. An isolated amino acid sequence encoding a CMP-N-Acetyneuraminic-Acid hydroxylase protein wherein the sequence is SEQ ID NO 4.

8. An isolated amino acid sequence encoding a CMP-N-Acetyneuraminic-Acid hydroxylase protein wherein the sequence is SEQ ID NO 6.

9. An isolated amino acid sequence encoding a CMP-N-Acetyneuraminic-Acid hydroxylase protein wherein the sequence is SEQ ID NO 8.

10. A nucleic acid construct comprising a full length cDNA sequence encoding a porcine CMP-N-Acetyneuraminic-Acid hydroxylase protein.

11. The construct of claim 10 wherein the cDNA is SEQ ID NO I.

12. The construct of claim 10 wherein the cDNA is SEQ ID NO 3.

13. The construct of claim 10 wherein the cDNA is SEQ ID NO 5.

14. The construct of claim 10 wherein the cDNA is SEQ ID NO 7.

15. The construct any one of claims 10-15 further comprising a promoter.

16. The construct of any one of claims 10-15 further comprising a selectable marker.

17. The construct of claim 16, wherein the selectable marker is green fluorescent protein.

18. A transfected cell comprising the construct of any one of claims 10-17.

19. A cell expressing a porcine CMP-N-Acetyneuraminic-Acid hydroxylase protein.

20. The cell of claim 19 wherein the protein sequence is selected from the group consisting of SEQ ID No 2, SEQ ID No 4, SEQ ID No 6 and SEQ ID No 8.

21. An isolated nucleotide sequence homologous to a nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

22. The nucleotide sequence of claim 21, wherein the sequence is at least 80% homologous to the nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

23. The nucleotide sequence of claim 21, wherein the sequence is at least 90% homologous to the nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

44. The nucleotide of claim 21, wherein the sequence is at least 90% homologous to a nucleotide sequence of any of claims 29 to 33.

24. A nucleic acid construct comprising a nucleotide sequence homologous to a nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

25. An isolated nucleotide sequence that hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

26. The nucleotide sequence of claim 25 that hybridizes under stringent conditions.

27. A vector comprising a nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

28. A plasmid comprising a nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7.

29. An isolated nucleotide comprising a nucleotide sequence of SEQ ID No 46.

42. An isolated nucleotide comprising a sequence homologous to a nucleotide sequence of any of claims 29 to 33.

43. The nucleotide of claim 41, wherein the sequence is at least 80% homologous to a nucleotide sequence of any of claims 29 to 33.

45. An isolated nucleotide that hybridizes to a nucleotide sequence of any of claims 29 to 33.

46. The nucleotide of claim 45 that hybridizes under stringent conditions.

30. An isolated nucleotide comprising a nucleotide sequence of SEQ ID No 47.

31. An isolated nucleotide comprising a nucleotide sequence of SEQ ID No 48.

34. A vector comprising a sequence of any of claims 31-33.

35. A nucleic acid construct comprising a sequence of any of claims 31-33.

36. A plasmid comprising a sequence of any of claims 31-33.

32. An isolated nucleotide sequence selected from the group consisting of SEQ ID No 10, SEQ ID No 1, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21, SEQ ID No 22, SEQ ID No 22, SEQ ID No 23, SEQ ID No 24, SEQ ID No 25, SEQ ID No 26, SEQ ID No 27 and SEQ ID No 28.

33. An isolated nucleotide sequence selected from the group consisting of SEQ ID No 9, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31, SEQ ID No 32, SEQ ID No 33, SEQ ID No 34, SEQ ID No 35, SEQ ID No 36, SEQ ID No 37, SEQ ID No 38, SEQ ID No 39, SEQ ID No 40, SEQ ID No 41, SEQ ID No 42, SEQ ID No 43, SEQ ID No 44 and SEQ ID No 45.

37. A nucleic acid construct comprising at least 17 contiguous nucleic acids of a sequence selected from the group comprising SEQ ID No 9, SEQ ID No 10, Seq ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 19, SEQ ID No 21, SEQ ID No 25, SEQ ID No 29, SEQ ID No 30, SEQ ID No31, SEQ ID No 32, SEQ ID No 33, SEQ ID No 39, SEQ ID No 40, SEQ ID No 41, SEQ ID No 42, SEQ ID No 43, SEQ ID No 44 and SEQ ID No 45.

38. A nucleic acid construct comprising at least 17 contiguous nucleic acids of SEQ ID No 47.

39. A nucleic acid construct comprising at least 2,775 contiguous nucleic acids of SEQ ID Nos 48.

40. A nucleic acid construct comprising at least 1,750 contiguous nucleic acids of SEQ ID Nos 49.

41. A nucleic acid construct comprising at least 150 contiguous nucleic acids of SEQ ID Nos 50.

47. A targeting vector comprising:

(a) a first nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 47;

(b) a selectable marker gene; and

(c) a second nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 48.

49. The targeting vector of claim 47 or 48 wherein the selectable marker is green fluorescent protein.

50. The targeting vector of claim 47 or 48 wherein the first nucleotide sequence represents the 5′

recombination arm.

51. The targeting vector of claim 47 or 48 wherein the second nucleotide sequence represents the 3′

recombination arm.

52. The targeting vector of claim 49 wherein the selectable marker is green fluorescent protein.

55. A cell transfected with the targeting vector of any one of claims 47 to 54.

56. The cell of claim 55 wherein at least one allele of a porcine CMP-N-Acetyeuraminic-Acid hydroxylase gene has been rendered inactive via homologous recombination.

57. A porcine animal comprising the cell of claim 55.

58. The animal of claim 57 wherein at least one allele of a porcine CMP-N-Acetyeuraminic-Acid hydroxylase gene has been rendered inactive via homologous resombination.

59. An organ obtained from the animal of claim 58.

60. A tissue obtained from the animal of claim 58.

61. The organ of claim 59 wherein the organ is selected from the group consisting of heart, lung, kidney and liver.

62. A method to produce a genetically modified cells comprising:

(a) transfecting a porcine cell with the targeting vector of claim 47 or 48; and

(b) selecting a transfected cell in which at least one allele of a porcine CMP-N-Acetyeuraminic-Acid hydroxylase gene has been rendered inactive.

63. A method to produce a genetically modified animal comprising:

(a) transfecting a porcine cell with the targeting vector of claim 47 or 48;

(b) selecting a tranfected cell in which at least one allele of a porcine CMP-N-Acetyeuraminic-Acid hydroxylase gene has been rendered inactive (a nuclear donor cell);

(c) transferring the nucleus of the nuclear donor cell into an enucleated oocyte to produce an embryo; and

(d) allowing the embryo to develop into an animal.

64. An organ derived from the animal of claim 63.

65. The organ of claim 64 wherein the organ is selected from the group consisting of heart, lung, kidney and liver.

66. A tissue derived from the animal of claim 63.

48. A targeting vector comprising:

(a) a first nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 48;

(b) a selectable marker gene; and

(c) a second nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 48;

wherein the first and second nucleotide sequences do not overlap.

53. The targeting vector of claim 48 wherein the first nucleotide sequence is homologous to a nucleotide sequence comprising at least 17 contiguous nucleotides of Intron 5.

54. The targeting vector of claim 48 wherein the second nucleotide sequence is homologous to a nucleotide sequence comprising at least 17 contiguous nucleotides of Intron 6.