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BREAKTHROUGH TECHNOLOGIES

Generation of Single-Copy T-DNA Transformants in Arabidopsis by the CRE/loxP Recombination-Mediated Resolution System¹

Department of Plant Systems Biology, Flanders Institute for Biotechnology, and Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium

We investigated whether complex T-DNA loci, often resulting in low transgene expression, can be resolved efficiently into single copies by CRE/loxP-mediated recombination. An SB-loxP T-DNA, containing two invertedly oriented loxP sequences located inside and immediately adjacent to the T-DNA border ends, was constructed. Regardless of the orientation and number of SB-loxP-derived T-DNAs integrated at one locus, recombination between the outermost loxP sequences in direct orientation should resolve multiple copies into a single T-DNA copy. Seven transformants with a complex SB-loxP locus were crossed with a CRE-expressing plant. In three hybrids, the complex T-DNA locus was reduced efficiently to a single-copy locus. Upon segregation of the CRE recombinase gene, only the simplified T-DNA locus was found in the progeny, demonstrating DNA had been excised efficiently in the progenitor cells of the gametes. In the two transformants with an inverted T-DNA repeat, the T-DNA resolution was accompanied by at least a 10-fold enhanced transgene expression. Therefore, the resolution of complex loci to a single-copy T-DNA insert by the CRE/loxP recombination system can become a valuable method for the production of elite transgenic Arabidopsis thaliana plants that are less prone to gene silencing.

² These authors contributed equally to the article.

³ Present address: CropDesign NV, a BASF Plant Science Company, Technologiepark 3, 9052 Gent, Belgium.

⁴ Present address: Institute for Agricultural and Fisheries Research, Unit Technology and Food, Ministry of the Flemish Community, Brusselsesteenweg 370, 9090 Melle, Belgium.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ann Depicker (ann.depicker{at}psb.ugent.be).

www.plantphysiol.org/cgi/doi/10.1104/pp.107.104067

^* Corresponding author; e-mail ann.depicker{at}psb.ugent.be.

Received June 19, 2007; accepted July 18, 2007; published August 10, 2007.

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