Porcine Isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region

• US 20050155095A1
• Filed: 11/05/2004
• Published: 07/14/2005
• Est. Priority Date: 11/05/2003
• 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 7,560,538 B2
• Time in Patent Office: Days
• US Class Current: 800/17
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/108 Swine
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12Y 204/01087 N-Acetyllactosaminide 3-alpha-galactosyltransferase (2.4.1.87), i.e. alpha-1,3-galactosyltransferase

Abstract

The present invention provides porcine isogloboside 3 (iGb3) synthase protein, cDNA, and genomic DNA regulatory sequence. The present also invention includes porcine animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which lack expression of functional iGb3 synthase. Such animals, tissues, organs and cells can be used in research and in medical therapy, including xenotransplantation. In addition, methods are provided to prepare organs, tissues, and cells lacking the porcine iGb₃ synthase gene for use in xenotransplantation.

Claims

1. An isolated cDNA sequence encoding a porcine iGb3 synthase peptide, comprising SEQ ID NO 1.

2. An isolated amino acid sequence encoding a iGb3 synthase peptide comprising SEQ ID NO2.

3. A nucleic acid construct comprising a cDNA sequence encoding a porcine iGb3 synthase protein comprising SEQ ID NO 1.

4. The construct of claim 3 further comprising a promoter.

5. The construct of claim 3 further comprising a selectable marker.

6. The construct of claim 5 wherein the selectable marker is green fluorescent protein.

7. A transfected cell comprising the construct of claim 3.

8. A cell expressing a porcine iGb3 protein sequence comprising SEQ ID NO 2.

9. An isolated nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 13.

10. A nucleic acid construct comprising at least 17 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 12, and SEQ ID No. 13.

11. A nucleic acid construct comprising at least 500 contiguous nucleic acids selected from the group consisting of SEQ ID No. 6 and SEQ ID No. 11.

13. A nucleic acid construct comprising a nucleic acid sequence encoding at least 160 contiguous amino acids of SEQ ID No. 2.

14. An isolated nucleotide comprising a sequence homologous to a nucleotide sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 12, and SEQ ID No. 13.

15. The nucleotide sequence of claim 14, wherein the sequence is at least 80% homologous.

16. The nucleotide sequence of claim 14, wherein the sequence is at least 90% homologous.

17. An isolated nucleotide comprising a sequence homologous to a nucleotide sequence selected from the group consisting of SEQ ID No. 6 and SEQ ID. No. 11.

20. An isolated nucleotide that hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 12, and SEQ ID No. 13.

21. The nucleotide of claim 20, wherein the sequence hybridizes under stringent conditions.

22. An isolated nucleotide that hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID No. 6 and SEQ ID No. 11.

23. The nucleotide of claim 22, wherein the sequence hybridizes under stringent conditions.

24. A targeting vector comprising:

a) a first nucleotide sequence comprising at least 500 contiguous nucleic acid sequences homologous to SEQ ID No. 11; and

b) a second nucleotide sequence comprising at least 500 contiguous nucleic acid sequences homologous to SEQ ID No. 11,

wherein the first and second nucleotide sequences do not overlap.

12. The nucleic acid construct of claim 31, wherein the construct comprises at least 1000 nucleic acids.

25. The targeting vector of claim 24, wherein the first nucleotide sequence represents a 5′

recombination arm.

26. The targeting vector of claim 24, wherein the second nucleotide sequence represents a 3′

recombination arm.

27. A cell comprising the vector of claim 24.

28. The cell of claim 27, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

29. A porcine animal comprising the cell of claim 27.

30. The animal of claim 29, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

31. An organ obtained from the animal of claim 29.

32. The organ of claim 31, wherein the organ is selected from the group consisting of heart, lung, kidney, and liver.

33. A tissue obtained from the animal of claim 29.

34. A targeting vector comprising:

a) a first nucleotide sequence comprising at least 17 contiguous nucleic acid sequences homologous to SEQ ID No. 12 or SEQ ID No. 13 and

b) a second nucleotide sequence comprising at least 17 contiguous nucleic acid sequences homologous to SEQ ID No. 12 or SEQ ID No. 13,

wherein the first and second nucleotide sequences do not overlap.

18. The nucleotide sequence of claim 37, wherein the sequence is at least 80% homologous.

19. The nucleotide sequence of claim 37, wherein the sequence is at least 90% homologous.

35. The targeting vector of claim 34, wherein the first nucleotide sequence represents a 5′

recombination arm.

36. The targeting vector of claim 34, wherein the second nucleotide sequence represents a 3′

recombination arm.

37. A cell comprising the targeting vector of claim 34.

38. The cell of claim 37, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

39. A porcine animal comprising the cell of claim 37.

40. The animal of claim 39, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

41. An organ obtained from the animal of claim 39.

42. The organ of claim 41, wherein the organ is selected from the group consisting of heart, lung, kidney, and liver.

43. A tissue obtained from the animal of claim 39.

44. A targeting vector comprising:

a) a first nucleotide sequence comprising at least 17 contiguous nucleic acid sequences homologous to the sequence selected form the group consisting of SEQ ID No. 3;

SEQ ID No. 4, SEQ ID No. 7. SEQ ID No. 8, and SEQ ID No. 9; and

b) a second nucleotide sequence comprising at least 17 contiguous nucleic acid sequences homologous to SEQ ID No. 10.

45. The targeting vector of claim 44, wherein the first nucleotide sequence represents a 5′

recombination arm.

46. The targeting vector of claim 44, wherein the second nucleotide sequence represents a 3′

recombination arm.

47. A cell comprising the vector of claim 44.

48. The cell of claim 47, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

49. A porcine animal comprising the cell of claim 47.

50. The animal of claim 49, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

51. An organ obtained from the animal of claim 49.

52. The organ of claim 51, wherein the organ is selected from the group consisting of heart, lung, kidney, and liver.

53. A tissue obtained from the animal of claim 49.

54. A targeting vector comprising:

a) a first nucleotide sequence comprising at least 17 contiguous nucleic acid sequences homologous to the sequence selected form the group consisting of SEQ ID No. 3;

SEQ ID No. 4, SEQ ID No. 7. SEQ ID No. 8, and SEQ ID No. 9; and

b) a second nucleotide sequence comprising at least 500 contiguous nucleic acid sequences homologous to the sequence selected from the group consisting of SEQ ID No. 6 and SEQ ID No. 10.

55. The targeting vector of claim 54, wherein the first nucleotide sequence represents a 5′

recombination arm.

56. The targeting vector of claim 54, wherein the second nucleotide sequence represents a 3′

recombination arm.

57. A cell comprising the vector of claim 56.

58. The cell of claim 57, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

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

60. The animal of claim 59, wherein at least one allele of a porcine iGb3 synthase gene has been rendered inactive via homologous recombination.

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

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

63. A tissue obtained from the animal of claim 59.