Agrobacterium‐mediated RMCE approach for gene replacement

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We describe the site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). The system requires the selection of a transformed line with an integrated copy of a target cassette, and subsequent introduction of an exchange vector. The target cassette contains the npt and cod genes between oppositely orientated recognition sites (RS). The exchange vector T-DNA possesses an exchange cassette containing the gene of interest and a selectable marker gene, such as hpt, between oppositely orientated (inner) RS. Adjacent to the exchange cassette are ipt and recombinase (R) genes and an additional (outer) RS. The recombinase catalyses double-crossover between target RS and exchange inner RS to replace the integrated target cassette with the introduced exchange cassette. Transgenic plants that contain randomly integrated copies of the exchange vector T-DNA show an abnormal phenotype as a result of the overproduction of cytokinin from ipt gene expression. The recombinase can also act on the directly orientated outer RS to remove such randomly integrated copies. The system resulted in single-copy exchange into the target site only in regenerated tobacco at a frequency of 1%-3% per treated explant, or 4%-9% per regenerated line of normal phenotype. Thus, transgenic plants with only an exchanged copy can be efficiently accumulated and selected. Here, we show that the SDI system can efficiently replace the target cassettes with the exchange cassettes in a heterozygous or homozygous condition. The SDI system may be useful for precise comparisons of different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.