Methods for the replacement, translocation and stacking of DNA in eukaryotic genomes

• US 6,936,747 B2
• Filed: 07/23/2001
• Issued: 08/30/2005
• Est. Priority Date: 07/21/2000
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Agriculture
• Inventors: Ow, David W.
• Primary Examiner(s): McElwain, Elizabeth F.
• Assistant Examiner(s): HELMER, GEORGIA L
• Application Number:
  US09/911,088
• Publication Number:
  US 20020123145A1
• Time in Patent Office: 1,499 Days
• Field of Search: 800/278, 800/288, 800/298, 800/306, 536/23.1, 435/320.1, 435/419
• US Class Current: 800/294
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome

Abstract

The present invention includes compositions and methods for site-specific polynucleotide replacement in eukaryotic cells. These methods include single polynucleotide replacement as well as gene stacking methods. Preferred eukaryotic cells for use in the present invention are plant cells and mammalian cells.

Claims

1. A method of obtaining site-specific replacement of a DNA of interest in a plant cell, comprising:

a) providing a plant cell that comprises a receptor construct, wherein the receptor construct comprises a receptor polynucleotide to be replaced, the receptor polynucleotide being flanked by two or more copies of a irreversible recombination site (IRS);

b) introducing into the cell a donor construct that comprises a donor polynucleotide to replace the receptor polynucleotide, the donor polynucleotide being flanked by two or more copies of a complementary irreversible recombinase site (CIRS), and

c) contacting the receptor construct and donor construct with an irreversible recombinase polypeptide;

wherein the irreversible recombinase catalyzes recombination between IRS and CIRS and replacement of the receptor polynucleotide with the donor polynucleotide, thereby forming a replacement construct.

2. The method of claim 1, wherein the donor construct is linear.

3. The method of claim 1, wherein the donor construct is a circular vector.

4. The method of claim 1, wherein the donor construct is a chromosome.

5. The method of claim 1, wherein the receptor construct is a chromosome.

6. The method of claim 1, wherein the receptor construct comprises two copies of the IRS and the donor construct comprises two copies of the CIRS.

7. The method of claim 6, wherein the IRS are inverted with respect to each other and wherein the CIRS are inverted with respect to each other.

8. The method of claim 6, wherein the donor polynucleotide further comprises a promoter operably linked to a DNA of interest.

9. The method of claim 6, wherein the receptor construct further comprises a promoter that is adjacent to one copy of the IRS.

10. The method of claim 9, wherein the promoter is located in the 5 prime direction from the IRS.

11. The method of claim 9, wherein the receptor construct further comprises a second promoter operably linked to a selectable marker.

12. The method of claim 9, wherein the receptor polynucleotide or the donor polynucleotide further comprises a negative selectable marker.

13. The method of claim 9, wherein the receptor polynucleotide or the donor polynucleotide further comprises a nucleic acid encoding the irreversible recombinase polypeptide.

14. The method of claim 13, wherein the receptor polynucleotide comprises the nucleic acid encoding the irreversible recombinase polypeptide.

15. The method of claim 14, wherein the irreversible recombinase is a bacteriophage φ

C31 integrase.

16. The method of claim 1, wherein the plant cell is an Arabidopsis thaliana cell.