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 replacement of a DNA of interest in a mammalian cell, comprising:
- a) providing a mammalian 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 of a complementary irreversible recombination site (CIRS); and
c) contacting the receptor construct and the donor construct with an irreversible recombinase polypeptide;
wherein the irreversible recombinase catalyzes recombination between the IRS and the CRIS 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
35 Claims
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1. A method of obtaining site-specific replacement of a DNA of interest in a mammalian cell, comprising:
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a) providing a mammalian 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 of a complementary irreversible recombination site (CIRS); and c) contacting the receptor construct and the donor construct with an irreversible recombinase polypeptide; wherein the irreversible recombinase catalyzes recombination between the IRS and the CRIS 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)
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Specification