Plant Molecular Biology

, Volume 35, Issue 4, pp 523–530 | Cite as

Gene targeting approaches using positive-negative selection and large flanking regions

  • Thomas Thykjær
  • Jørgen Finnemann
  • Leif Schauser
  • Liselotte Christensen
  • Carsten Poulsen
  • Jens Stougaard*


We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli. Gene targeting was approached on the Gln1 and the Pzf loci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors. Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination. For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over. A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events. The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection. Using five different replacement vectors an estimated total of 18974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained. Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis. With this approach the frequency of detectable gene targeting in L. japonicus was below 5.3×10−5, despite the large flanking sequences offered for recombination.

cytosine deaminase homologous recombination Lotus japonicus negative selection plants 


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Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Thomas Thykjær
    • 1
  • Jørgen Finnemann
    • 2
  • Leif Schauser
    • 1
  • Liselotte Christensen
    • 1
  • Carsten Poulsen
    • 1
  • Jens Stougaard*
    • 1
  1. 1.Laboratory of Gene Expression, Department of Molecular and Structural BiologyUniversity of AarhusAarhus CDenmark
  2. 2.Plant Nutrition Laboratory, Dep. of Agricultural SciencesThe Royal Veterinary and Agricultural UniversityCopenhagenDenmark

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