title: Enrichments of gene replacement events byAgrobacterium-mediated recombinase-mediated cassette exchange.
abstract: We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by usingAgrobacterium-mediated transformation. Transgenic tobacco plants which have a single copy of negative marker genes (codA) at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method. By negative selection, we were able to obtain five heterozygous and four homozygous transgenic plants in which the genes were exchanged from 64 leaf segments of heterozygous and homozygous target plants, respectively. Except for one transgenic plant with an extra copy, the other eight plants had only a single copy of marker-free transgenes, and no footprint of random integrated copies was detected in half of the eight plants. The RMCE re-transformation frequencies were calculated as 6.25 % per explant and were approximately the same as the average percentage of intact single-copy transformation events for standard tobaccoAgrobacterium-mediated transformation.
authors.affiliation: Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano, 386-8567 Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Ebinuma
authors.affiliation: Agri-Biotechnology Research Laboratory, Nippon Paper Industries Co. Ltd., 5-21-1, Oji, Kita-ku, Tokyo, 114-0002 Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Nakahama
authors.affiliation: Agri-Biotechnology Research Laboratory, Nippon Paper Industries Co. Ltd., 5-21-1, Oji, Kita-ku, Tokyo, 114-0002 Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nanto
keywords: Gene replacement; Negative selection; RMCE