Generation of single-copy T-DNA transformants in Arabidopsis by the CRE/loxP recombination-mediated resolution system.
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.
De Buck
Sylvie
S
Department of Plant Systems Biology, Flanders Institute for Biotechnology, and Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium.
Peck
Ingrid
I
De Wilde
Chris
C
Marjanac
Gordana
G
Nolf
Jonah
J
De Paepe
Annelies
A
Depicker
Ann
A
D017360:Arabidopsis / Q000235:genetics*; D004269:DNA, Bacterial; D018628:Gene Dosage; D015870:Gene Expression; D019426:Integrases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014170:Transformation, Genetic; D019076:Transgenes
D004269:DNA, Bacterial; D012333:RNA, Messenger; C036483:T-DNA; C045073:Cre recombinase; D019426:Integrases
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