Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event

Cre recombinase was used to mediate recombination between a chromosomally i ntroduced loxP sequence in Arabidopsis thaliana (35S-lox-cre) and transferr ed DNA (T-DNA) originating from Agrobacterium tumefaciens (plox-npt), calrr ying a single loxP sequence. Constructs were designed For specific Cre-medi ated rt combination between the two lox sites, resulting in restoration of neomycin phosphotransferase (nptII) expression at the target locus. Kanamyc in resistant (Km(r)) recombinants were obtained with an efficiency of about 1%, compared with random integration. Molecular analyses confirmed that th ese were indeed due to recombination between the los sites of the target an d introduced T-DNA. However, polymerase chain reaction analysis revealed th at these reflected site-specific integration events only in a minority (4%) . The other events were classified as translocations/inversions (71%) or de letions (25%), and were probably caused by site-specific recombination betw een a randomly integrated T-DNA and the original target locus. We studied s ome of these events in detail, including a Cre-mediated balanced translocat ion event, which was characterized by a combination of molecular, genetic a nd cytogenetic experiments (fluorescence in situ hybridization to spread po llen mother cells at meiotic prophase I). Our data clearly demonstrate that Agrobacterium-mediated transfer of a targeting T-DNA with a single (ox sit e allows the isolation of multiple chromosomal rearrangements, including tr anslocation and deletion events. Given that the complete sequence of the Ar abidopsis genome will have been determined shortly this method has signific ant potential for applications in functional genomics.