Claims
- 1. A method for transferring genetic material, which is not a typical T-DNA surrounded by Agrobacterium T-borders from an Agrobacterium virulence system, to a eukaryotic host cell, said method comprising:
providing said genetic material on a mobilizable plasmid capable of forming a relaxosome; bringing said mobilizable plasmid into an Agrobacterium having at least the activity of the transfer genes of Agrobacterium not present on said mobilizable plasmid so that the necessary gene products providing the same or similar activity as a functional VirB operon are also present; and co-cultivating said Agrobacterium with the eukaryotic host cell.
- 2. The method according to claim 1, wherein said mobilizable plasmid comprises:
a functional oriT and a sequence encoding VirD-like mobilization activity as well as a sequence encoding a VirD4-like coupling factor.
- 3. The method according to claim 1, wherein said mobilizable plasmid comprises a functional oriT, but wherein VirD-like mobilization functions and the VirD4-like coupling factor are provided in trans.
- 4. The method according to claim 2, wherein said mobilizable plasmid comprises a functional oriT, but wherein VirD-like mobilization functions and the VirD4-like coupling factor are provided in trans.
- 5. The method according to claim 1, claim 2, claim 3 or claim 4, wherein said mobilizable plasmid is derived from a group of non-conjugative mobilizable plasmids present in enterobacteria.
- 6. The method according to claim 5, wherein said group of mobilizable plasmids comprises small plasmids which can be maintained in high copy number in enterobacteria.
- 7. The method according to claim 6, wherein said group of enterobacteria comprises E. coli.
- 8. The method according to any one of the previous claims, wherein said mobilizable plasmid is derived from CloDF13.
- 9. The method according to any one of the previous claims wherein said mobilizable plasmid is produced and or multiplied in an enterobacterium.
- 10. The method according to claim 9, wherein said enterobacterium is E. coli.
- 11. A mobilizable plasmid comprising:
genetic material to be transferred into a eukaryotic cell by Agrobacterium transfer; a functional oriT; sequences encoding functional virD-like mobilization products; a VirD4-like coupling factor; and sequences encoding functional virB-like activity.
- 12. The mobilizable plasmid of claim 11, which encodes the Mob functions of CloDF13.
- 13. The mobilizable plasmid of claim 11, wherein said mobilizable plasmid is derived from a group of non-self-conjugative mobilizable plasmids present in enterobacteria.
- 14. The mobilizable plasmid of claim 12, wherein said mobilizable plasmid is derived from a group of non-self-conjugative mobilizable plasmids present in enterobacteria.
- 15. The mobilizable plasmid of claim 13, wherein said mobilizable plasmid is derived from CloDF13.
- 16. The mobilizable plasmid of claim 14, wherein said mobilizable plasmid is derived from CloDF13.
- 17. In a method of transferring genetic material to a eukaryotic cell of the type wherein a plasmid is used to transfer the genetic material, the improvement comprising:
using the mobilizable plasmid of any one of claims 11-14 for the transfer of genetic material to the eukaryotic cell.
- 18. The improvement of claim 17, wherein said transfer is to the nucleus or an organelle of the eukaryotic cell.
- 19. The improvement of claim 17, wherein said eukaryotic cell is selected from the group consisting of a plant cell, a yeast cell, and a fungal cell.
- 20. The improvement of claim 18, wherein said eukaryotic cell is selected from the group consisting of a plant cell, a yeast cell, and a fungal cell.
Priority Claims (1)
Number |
Date |
Country |
Kind |
00200726.8 |
Mar 2000 |
EP |
|
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of pending PCT International Patent Application Number PCT/NL01/00169, filed Mar. 1, 2001, (published in English as WO 01/64925 A1 on Sep. 7, 2001, the contents of which are incorporated by this reference).
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
PCT/NL01/00169 |
Mar 2001 |
US |
Child |
10234048 |
Sep 2002 |
US |