Compositions and methods to reduce the complexity of transgene integration into the genome of a plant

• US 20030237107A1
• Filed: 05/07/2003
• Published: 12/25/2003
• Est. Priority Date: 11/18/1997
• Status: Active Grant

• Current Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Original Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Inventors: Baszczynski, Christopher L., Bowen, Benjamin A., Peterson, David J., Tagliani, Laura
• Granted Patent: US 7,405,079 B2
• Time in Patent Office: Days
• US Class Current: 800/260
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/8203 Virus mediated transformation
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

Methods for the targeted integration of nucleotide sequences into a plant are provided. Transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are used to transform a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase.

Claims

1. A method for reducing the complexity of integration of transgenes in a genome of a plant cell, said method comprising:

a) introducing into the plant cell a transfer cassette flanked by or comprising a first and a second recombination site, wherein said first and said second recombination sites are non-identical, wherein said transfer cassette further comprises a nucleotide sequence of interest and, wherein said genome of the plant cell comprise a target site comprising the first and the second non-identical recombination sites of said transfer cassette;

b) providing a recombinase that recognizes and implements recombination at the non-identical recombination sites; and

,

c) selecting the plant cells having reduced complexity of integrated transgenes in their genome, wherein said reduced complexity comprises the integration of the transfer cassette at the target site.

2. The method of claim 1, wherein introducing into the plant cell a transfer cassette comprises sexual breeding.

3. The method of claim 1, wherein providing the recombinase comprises integrating in the genome of said plant cells a nucleotide sequence encoding said recombinase.

4. The method of claim 1, wherein said first or said second non-identical recombination site is selected from the group consisting of an FRT site, a mutant FRT site, a LOX site, and a mutant LOX site.

5. The method of claim 4, wherein said first or said second non-identical recombination site is selected from the group consisting of the FRT site and the mutant FRT site.

6. The method of claim 1, wherein providing said recombinase comprises genetically transforming said plant cells with an expression cassette comprising a nucleotide sequence encoding said recombinase.

7. The method of claim 1, wherein said recombinase is FLP.

8. The method of claim 7, wherein said FLP has been synthesized using maize preferred codons.

9. The method of claim 5, wherein said mutant FRT site is FRT 5 (SEQ ID NO:

3), FRT 6 (SEQ ID NO;

     4), or FRT 7 (SEQ ID NO;

     5).

10. The method of claim 1, wherein said plant cell is from a monocot.

11. The method of claim 10, wherein said monocot is maize.

12. The method of claim 1, wherein said plant cell is from a dicot.

13. The method of claim 12, wherein said dicot is canola, Brassica, soybean, sunflower, or cotton.

14. The method of claim 1, wherein said recombinase is Cre.

15. The method of claim 14, wherein said Cre recombinase has been synthesized using maize preferred codons.

16. The method of claim 1, wherein the plant cells having reduced complexity of integrated transgenes comprise plant cells having the transfer cassette integrated at the target site and having a targeting frequency of the transfer cassette to the target site relative to total genomic integration of the transfer cassette between 1.5% to 66.7%.

17. The method of claim 16, wherein the targeting frequency of the transfer cassette to the target site relative to total genomic integration of the transfer cassette is between 10% to 20%.

18. The method of claim 16, wherein the targeting frequency of the transfer cassette to the target site relative to total genomic integration of the transfer cassette is between 20% to 66.7%.

19. The method of claim 16, wherein the targeting frequency of the transfer cassette to the target site relative to total genomic integration of the transfer cassette is between 1.5% to 10%.

20. The method of claim 1, wherein said plant cells are in a plant.

21. The method of claim 20, wherein said plant is a monocot.

22. The method of claim 21, wherein said monocot is maize.

23. The method of claim 20, wherein said plant is a dicot.