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AssigneeE. I. Du Pont de Nemours and Company
InventorsSaverio Carl Falco, Zhongsen Li
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A targeting method is described that allows precise cassette replacement at a previously characterized genetic locus. A target DNA construct containing a pair of incompatible FRT sites flanking a target cassette was introduced into soybean by regular biolistic transformation. Transgenic events containing a single complete copy of the target site were then selected and retransformed with a donor DNA construct containing the identical pair of incompatible FRT sites flanking a donor cassette. Precise DNA cassette exchange happened between the target cassette and the donor cassette via recombinase mediated cassette exchange (RMCE) so that the donor cassette was introduced at the exact genomic site previously occupied by the target cassette. Through repeated RMCE using additional incompatible FRT sites, multiple groups of transgenes can be stacked at the same genomic locus.
A method for stacking multiple expression cassettes of interest into a specific chromosomal site in a soybean genome, said method comprising:
A soybean cell, plant or seed having stably incorporated in its genome a transfer cassette comprising at least three non-identical recombination sites, where the transfer cassette comprises a polynucleotide encoding a selectable marker protein-coding sequence bounded by a first recombination site and a second non-identical recombination site, wherein the transfer cassette further comprises a third non-identical recombination site bounded by the selectable marker protein-coding sequence and the second non-identical recombination site, wherein the transfer cassette further comprises at least one expression cassette of interest, wherein the at least one expression cassette of interest is bounded by the third non-identical recombination site and the second non-identical recombination site.
A method for stacking of multiple expression cassettes of interest into a specific chromosomal site in a soybean genome, said method comprising:
A method for creating a transgenic soybean cell comprising a target site suitable for stacking of multiple expression cassettes of interest into a specific chromosomal site in a soybean genome, said method comprising transforming a soybean cell with an isolated nucleic acid fragment comprising at least one first expression cassette of interest adjacent to a target site, wherein said target site comprises a selectable marker protein-coding sequence, wherein the selectable marker protein-coding sequence is bounded by a first recombination site and a second non-identical recombination site.
A soybean cell, plant or seed having stably incorporated in its genome an isolated nucleic acid fragment comprising at least one first expression cassette of interest adjacent to a target site, wherein said target site comprises a selectable marker protein-coding sequence, wherein the selectable marker protein-coding sequence is bounded by a first recombination site and a second non-identical recombination site.
A soybean cell, plant or seed having stably incorporated in its genome an isolated nucleic acid fragment comprising a target site, wherein said target site comprises a promoter operably linked to a selectable marker protein-coding sequence, wherein the selectable marker protein-coding sequence is bounded by a first recombination site and a second non-identical recombination site, further wherein the first recombination site is between the promoter and the selectable marker protein-coding sequence.
Dependencies are inferred from the claim text.