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Chromosoma

, Volume 109, Issue 4, pp 287–297 | Cite as

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

  • Annette C. Vergunst
  • Lars E.T. Jansen
  • Paul F. Fransz
  • J. Hans de Jong
  • Paul J.J. Hooykaas

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.

Keywords

Target Locus Polymerase Chain Reaction Analysis Pollen Mother Cell Meiotic Prophase Neomycin Phosphotransferase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2000

Authors and Affiliations

  • Annette C. Vergunst
    • 1
  • Lars E.T. Jansen
    • 1
  • Paul F. Fransz
    • 2
  • J. Hans de Jong
    • 2
  • Paul J.J. Hooykaas
    • 1
  1. 1.Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The NetherlandsNL
  2. 2.Laboratory of Genetics, Wageningen University, Wageningen, The NetherlandsNL

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