Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event |
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Authors: | Annette C Vergunst Lars ET Jansen Paul F Fransz J Hans de Jong Paul JJ Hooykaas |
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Institution: | (1) Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands, NL;(2) Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands, NL |
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Abstract: | Cre recombinase was used to mediate recombination between a chromosomally introduced loxP sequence in Arabidopsis thaliana (35S-lox-cre) and transferred DNA (T-DNA) originating from Agrobacterium tumefaciens (plox-npt), carrying a single loxP sequence. Constructs were designed for specific Cre-mediated recombination between the two lox sites, resulting in restoration of neomycin phosphotransferase (nptII) expression at the target locus. Kanamycin resistant (Kmr) recombinants were obtained with an efficiency of about 1% compared with random integration. Molecular analyses confirmed
that these were indeed due to recombination 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 recombination 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 molecular, genetic and cytogenetic experiments (fluorescence 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 applications in functional genomics.
Received: 29 December 1999; in revised form: 21 February 2000 / Accepted: 2 March 2000 |
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