Adenoviral fiber exchange shuttle system

1. A method for generating a recombinant adenoviral vector encoding a desired product, comprising the step(s) of co-transforming a cell expressing an enzyme that mediates homologous recombination with

a) a linearized shuttle plasmid encoding a selectable marker, and

b) a transfer plasmid, wherein the transfer plasmid comprises a fiber gene, and wherein the fiber gene is modified to contain a unique restriction site in the HI loop;

thereby allowing recombination of the plasmids to generate the recombinant adenoviral vector.

2. The method of claim 1 , wherein the enzyme that mediates homologous recombination is RecA.

3. The method of claim 1 , wherein the transfer plasmid is constructed by co-transforming into a cell expressing Cre recombinase

a) a donor plasmid, wherein the donor plasmid encodes the fiber gene flanked by lox sites and a second selectable marker, and

b) an acceptor plasmid, wherein the acceptor plasmid encodes a negatively selectable marker flanked by lox sites and a first selectable marker, and wherein the acceptor plasmid does not encode the fiber gene;

thereby allowing for recombination of the fiber gene and the negatively selectable marker.

4. The method of claim 3 , wherein the lox sites are incompatible.

5. The method of claim 3 , wherein the lox sites are mutated to result in unidirectional recombination.

6. The method of claim 3 , wherein the negatively selectable marker is SacB.

7. The method of claim 1 , wherein the unique restriction site is a BspEl site.

8. The method of claim 3 , wherein the donor plasmid lox sites are Lox m2/66 and Lox 71.

9. The method of claim 3 , wherein the acceptor plasmid lox sites are Lox m2/71 and Lox 66.

10. The method of claim 3 , wherein the acceptor plasmid contains a kanamycin selectable marker.

11. The method of claim 3 , wherein the donor plasmid contains an ampicillin selectable marker.

12. The method of claim 1 , wherein the method further comprises selecting recombinant adenoviral vectors using the selectable marker.

13. The method of claim 12 , wherein the selectable marker is kanamycin.

14. The method of claim 1 , wherein the cell is a bacterial cell.

15. The method of claim 14 , wherein the bacterial cell is an E. coli cell.

16. The method of claim 3 , wherein the cell is a bacterial cell.

17. The method of claim 16 , wherein the bacterial cell is an E. coli cell.

18. The method of claim 3 , wherein the cell is a mammalian cell.

19. The method of claim 1 , wherein the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding the desired product.

20. The method of claim 19 , wherein the product is selected from the group consisting of a polypeptide, polypeptides, or fragments thereof, a nucleic acid, an aptamer, an RNAi, an siRNA, and an shRNA.

21. The method of claim 20 , wherein the desired product is a polypeptide.

22. The method of claim 21 , wherein the desired polypeptide is a therapeutic polypeptide.

23. The method of claim 19 , wherein the nucleic acid segment is under control of a promoter.

24. The method of claim 23 , wherein the promoter is a tissue specific promoter.

25. The method of claim 19 , wherein the transfer plasmid comprises a resistance gene, and wherein the resistance gene in the shuttle plasmid is not the same as the resistance gene in the transfer plasmid.

26. The method of claim 2 , wherein the shuttle plasmid contains a unique restriction site located between RecA recombination sites.

27. The method of claim 26 , wherein the unique restriction site in the shuttle plasmid is a Pme I site.

28. The method of claim 1 , wherein the shuttle plasmid is linearized with Pme I.

29. The method of claim 1 , wherein the shuttle plasmid further comprises homologous recombination sites.

30. The method of claim 29 , wherein the homologous recombination sites are RecA homologous recombination sites.

31. The method of claim 30 , wherein the RecA homologous recombination sites are Ad5 left and Ad5 right.

32. The method of claim 1 , wherein the transfer plasmid further comprises homologous recombination sites.

33. The method of claim 32 , wherein the homologous recombination sites are RecA homologous recombination sites.

34. The method of claim 33 , wherein the RecA homologous recombination sites are Ad5 left and Ad5 right.

35. A method for generating a recombinant adenoviral vector encoding a desired gene product, the method comprising the step(s) of co-transforming a cell expressing Cre recombinase with

a) a donor plasmid, wherein the donor plasmid encodes a fiber gene flanked by lox sites, wherein the fiber gene is modified to contain a unique restriction site in the HI loop, and

b) a shuttle-acceptor plasmid encoding a negatively selectable marker, wherein the shuttle-acceptor plasmid does not encode the fiber gene;

thereby allowing for recombination of the fiber gene and the negatively selectable marker to generate the recombinant adenoviral vector.

36. The method of claim 35 , wherein the shuttle-acceptor plasmid encoding the negatively selectable marker is constructed by co-transforming a cell expressing an enzyme that mediates homologous recombination with

a) an acceptor plasmid, wherein the acceptor plasmid encodes the negatively selectable marker flanked by lox sites and a first selectable marker, and

b) a linearized shuttle plasmid encoding a second selectable marker,

thereby allowing for recombination of the first selectable marker and the second selectable marker to generate the shuttle-acceptor plasmid encoding the negatively selectable marker.

37. The method of claim 36 , wherein the enzyme that mediates homologous recombination is RecA.

38. The method of claim 35 , wherein the lox sites are incompatible.

39. The method of claim 35 , wherein the lox sites are mutated to result in unidirectional recombination.

40. The method of claim 35 , wherein the negatively selectable marker is SacB.

41. The method of claim 35 , wherein the unique restriction site is a BspEl site.

42. The method of claim 35 , wherein the donor plasmid lox sites are Lox m2/66 and Lox 71.

43. The method of claim 36 , wherein the acceptor plasmid lox sites are Lox m2/71 and Lox 66.

44. The method of claim 36 , wherein the acceptor plasmid contains a kanamycin selectable marker.

45. The method of claim 35 , wherein the donor plasmid contains an ampicillin selectable marker.

46. The method of claim 36 , wherein the method further comprises selecting recombinant adenoviral vectors using the second selectable marker or the negatively selectable marker.

47. The method of claim 46 , wherein the second selectable marker is kanamycin and the negatively selectable marker is SacB.

48. The method of claim 35 , wherein the cell is a bacterial cell.

49. The method of claim 48 , wherein the bacterial cell is an E. coli cell.

50. The method of claim 36 , wherein the cell is a bacterial cell.

51. The method of claim 50 , wherein the bacterial cell is an E. coli cell.

52. The method of claim 35 , wherein the cell is a mammalian cell.

53. The method of claim 36 , wherein the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding the desired product.

54. The method of claim 53 , wherein the desired product is selected from the group consisting of a polypeptide, polypeptides, or fragments thereof, a nucleic acid, an aptamer, an RNAi, an siRNA, and an shRNA.

55. The method of claim 54 , wherein the desired product is a polypeptide.

56. The method of claim 55 , wherein the desired polypeptide is a therapeutic polypeptide.

57. The method of claim 53 , wherein the nucleic acid segment is under control of a promoter.

58. The method of claim 57 , wherein the promoter is a tissue specific promoter.

59. The method of claim 37 , wherein the shuttle plasmid contains a unique restriction site located between RecA recombination sites.

60. The method of claim 59 , wherein the unique restriction site in the shuttle plasmid is a Pme I site.

61. The method of claim 36 , wherein the shuttle plasmid is linearized with Pme I.