Abstract. Cre recombinase was used to mediate recom- bination between a chromosomally introduced loxP se- quence in Arabidopsis thaliana (35S-lox-cre) and trans- ferred DNA (T-DNA) originating from Agrobacterium tumefaciens (plox-npt), carrying a single loxP sequence. Constructs were designed for specific Cre-mediated re- combination between the two lox sites, resulting in res- toration of neomycin phosphotransferase (nptII) expres- sion at the target locus. Kanamycin resistant (Km r ) re- combinants were obtained with an efficiency of about 1% compared with random integration. Molecular ana- lyses confirmed that these were indeed due to recombi- nation between the lox sites of the target and introduced T-DNA. However, polymerase chain reaction analysis revealed that these reflected site-specific integration events only in a minority (4%). The other events were classified as translocations/inversions (71%) or deletions (25%), and were probably caused by site-specific recom- bination between a randomly integrated T-DNA and the original target locus. We studied some of these events in detail, including a Cre-mediated balanced translocation event, which was characterized by a combination of mo- lecular, genetic and cytogenetic experiments (fluores- cence in situ hybridization to spread pollen mother cells at meiotic prophase I). Our data clearly demonstrate that Agrobacterium-mediated transfer of a targeting T-DNA with a single lox site allows the isolation of multiple chromosomal rearrangements, including translocation and deletion events. Given that the complete sequence of the Arabidopsis genome will have been determined shortly this method has significant potential for applica- tions in functional genomics. Introduction Site-specific recombination systems from prokaryotes and yeast, such as the bacteriophage P1 Cre/lox system, provide a powerful tool to create precise, controlled ge- nomic rearrangements in higher eukaryotes (Kilby et al. 1993; Van Haaren and Ow 1993; Ow and Medberry 1995; Kühn and Schwenk 1997). Cre recombinase medi- ates recombination between two 34 bp DNA recognition sequences (lox) without requirement for additional fac- tors or any host recombination enzymes. The outcome of a recombination reaction depends on the relative orienta- tion of two lox sites towards each other. Intramolecular recombination between two lox sites in direct orientation will generate a deletion of intervening DNA sequences, whereas an inverted orientation results in inversion. The presence of two lox sites on separate DNA molecules can result in co-integration in the case of circular mole- cules, or translocation-like events in the case of linear DNA molecules. In plants, such induced rearrangements have been used for the production of marker-free transgenic plants (Dale and Ow 1991; Gleave et al. 1999), creation of chromo- somal deletions or inversions (Medberry et al. 1995; Osborne et al. 1995), isolation of translocations (Qin et al. 1994), reduction of transgene copy number of complex loci (Srivastava et al. 1999), and site-specific integration of transgenes (Albert et al. 1995; Vergunst and Hooykaas 1998; Vergunst et al. 1998a). Furthermore, the possibility of engineering chromosomal rearrangements between pre- selected sites provides a powerful tool for studying the re- lation between genomic organization and function, such as the effect of chromosomal position on gene expression (Henikoff 1992; Golic and Golic 1996). To enable engineering of the plant genome, efficient transformation protocols are required. A widely used method for the production of transgenic plants makes use of the transfer system of Agrobacterium tumefaci- ens (Hooykaas and Beijersbergen 1994; Zupan and Zambryski 1997; Hansen and Chilton 1999). During the Agrobacterium infection process a segment of its Present address: P.F. Fransz, Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany Edited by: D. Schweizer Correspondence to: A.C. Vergunst e-mail: vergunst@rulbim.leidenuniv.nl Chromosoma (2000) 109:287–297 Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event Annette C. Vergunst 1 , Lars E.T. Jansen 1 , Paul F. Fransz 2 , J. Hans de Jong 2 , Paul J.J. Hooykaas 1 1 Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands 2 Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands Received: 29 December 1999; in revised form: 21 February 2000 / Accepted: 2 March 2000 © Springer-Verlag 2000