Methods for the replacement, translocation and stacking of DNA in eukaryotic genomes
First Claim
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1. A method of obtaining site-specific gene replacement in a eukaryotic cell, comprising:
- a) providing a eukaryotic cell that comprises a receptor construct, wherein the receptor construct comprises a receptor polynucleotide flanked by two or more of a irreversible recombination site (IRS);
b) introducing into the cell a donor construct that comprises a donor polynucleotide flanked by two or more of a irreversible complementary recombination site (CIRS); and
c) contacting the receptor construct and the donor construct with an irreversible recombinase polypeptide;
d) wherein the irreversible recombinase catalyzes recombination between the first and second types of recombination sites and replacement of the receptor polynucleotide with the donor polynucleotide, thereby forming a replacement construct.
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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.
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Citations
66 Claims
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1. A method of obtaining site-specific gene replacement in a eukaryotic cell, comprising:
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a) providing a eukaryotic cell that comprises a receptor construct, wherein the receptor construct comprises a receptor polynucleotide flanked by two or more of a irreversible recombination site (IRS);
b) introducing into the cell a donor construct that comprises a donor polynucleotide flanked by two or more of a irreversible complementary recombination site (CIRS); and
c) contacting the receptor construct and the donor construct with an irreversible recombinase polypeptide;
d) wherein the irreversible recombinase catalyzes recombination between the first and second types of recombination sites and replacement of the receptor polynucleotide with the donor polynucleotide, thereby forming a replacement construct. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A method of producing a transgenic plant, comprising the steps of:
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a) providing a receptor plant comprising a chromosomal receptor polynucleotide flanked by two of a irreversible recombination site (IRS);
b) providing a donor plant comprising a chromosomal donor polynucleotide flanked by two of a complementary irreversible recombination site (CIRS);
c) crossing the donor plant the receptor plant to produce a transgenic plant, wherein the transgenic plant expresses an irreversible recombinase polypeptide that catalyzes recombination between the IRS and the CIRS and replacement of the receptor polynucleotide with the donor polynucleotide, thereby forming a chromosomal replacement construct in the transgenic plant. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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53. A method of gene stacking in a cell comprising:
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a) providing a cell that comprises a target construct in a chromosome, wherein the target construct comprises a target polynucleotide, two of a reversible recombination site (RRS), wherein the RRS are oppositely oriented with respect to each other and wherein the target polynucleotide comprises at least one irreversible recombination site (IRS);
b) introducing into the cell a first donor construct that comprises a first donor polynucleotide, two of a complementary irreversible recombination site (CIRS) and two RRS that are oppositely oriented with respect to each other;
c) contacting the target construct and the first donor construct with an irreversible recombinase polypeptide that is compatible with each of the IRS and the CIRS, wherein the irreversible recombinase integrates the first donor polynucleotide into the target construct, thereby forming a first chromosomal integration construct;
d) deleting undesired nucleotide sequences in the first chromosomal integration construct by contacting the locus with a reversible recombinase polypeptide compatible with each of the RRS, thereby forming a first chromosomal trait construct;
e) introducing into the cell a second donor construct that comprises two IRS, a second donor polynucleotide and one RRS;
f) contacting the first chromosomal trait construct and the second donor construct with the irreversible recombinase polypeptide, wherein the irreversible recombinase integrates the second donor polynucleotide into the first chromosomal trait construct, thereby forming a second chromosomal integration construct;
g) selecting for a cell containing a second chromosomal integration construct wherein the first donor polynucleotide is adjacent to the second donor polynucleotide;
h) deleting undesired nucleotide sequences in the selected second chromosomal integration construct by contacting the selected second chromosomal integration construct with a reversible recombinase polypeptide compatible with each of the RRS, thereby forming a second chromosomal trait construct;
i) introducing into the cell a third donor construct that comprises two CIRS, a third donor polynucleotide and one RRS;
j) contacting the second chromosomal trait construct and the third donor construct with the irreversible recombinase polypeptide, wherein the irreversible recombinase integrates the third donor polynucleotide into the second chromosomal trait construct, thereby forming a third chromosomal integration construct;
k) selecting for a cell containing a third chromosomal integration construct wherein the second donor polynucleotide is adjacent to the third donor polynucleotide; and
l) deleting undesired nucleotide sequences in the selected third chromosomal integration construct by contacting the selected third chromosomal integration construct with a reversible recombinase polypeptide compatible with each of the RRS, thereby forming a third chromosomal trait construct. - View Dependent Claims (54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66)
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Specification