Nucleic Acids Research Advance Access published online on November 7, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl832
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Molecular Biology |
The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration
1 Centre for Plant Sciences, Faculty of Biological Sciences, Leeds University Leeds LS2 9JT, UK 2 Plant Biotechnology, Faculty of Biology, University of Freiburg Schaenzlestrasse 1, D-79104 Freiburg, Germany 3 Department of Biology, Washington University in St Louis St Louis, MO 63130-4899, USA 4 Forest Genetics, Department of Plant Sciences, Life Science Center Weihenstephan, Technische Universität München Am Hochanger 13, D-85354 Freising, Germany
*To whom correspondence should be addressed. Tel: +44 113 3433096; Fax: +44 113 3433144; Email: a.c.cuming{at}leeds.ac.uk
Received July 21, 2006. Revised September 25, 2006. Accepted October 8, 2006.
The model bryophyte Physcomitrella patens exhibits high frequencies of gene targeting when transformed with DNA constructs containing sequences homologous with genomic loci. ‘Targeted gene replacement’ (TGR) resulting from homologous recombination (HR) between each end of a targeting construct and the targeted locus occurs when either single or multiple targeting vectors are delivered. In the latter instance simultaneous, multiple, independent integration of different transgenes occurs at the targeted loci. In both single gene and ‘batch’ transformations, DNA can also be found to undergo ‘targeted insertion’ (TI), integrating at one end of the targeted locus by HR with one flanking sequence of the vector accompanied by an apparent non-homologous end-joining (NHEJ) event at the other. Untargeted integration at nonhomologous sites also occurs, but at a lower frequency. Molecular analysis of TI at a single locus shows that this occurs as a consequence of concatenation of the transforming DNA, in planta, prior to integration, followed by HR between a single site in the genomic target and two of its repeated homologues in the concatenated vector. This reinforces the view that HR is the major pathway by which transforming DNA is integrated in Physcomitrella.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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