title: In planta gene targeting.
abstract: The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a site-specific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.
authors.affiliation: Botany II,  Karlsruhe Institute of Technology, D-76131 Karlsruhe,  Germany.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Fauser
authors.forename: Nadine
authors.initials: N
authors.lastname: Roth
authors.forename: Michael
authors.initials: M
authors.lastname: Pacher
authors.forename: Gabriele
authors.initials: G
authors.lastname: Ilg
authors.forename: Rocío
authors.initials: R
authors.lastname: Sánchez-Fernández
authors.forename: Christian
authors.initials: C
authors.lastname: Biesgen
authors.forename: Holger
authors.initials: H
authors.lastname: Puchta
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D010944:Plants / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011995:Recombination, Genetic; D036226:Seedlings / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; D005966:Glucuronidase