Published online 10 November 2005
Methods Online |
Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens
Centre for Plant Sciences, Faculty of Biological Sciences, Leeds University Leeds LS2 9JT, UK
*To whom correspondence should be addressed. Tel: +44 113 3433096; Fax: +44 113 3433144; Email: a.c.cuming{at}leeds.ac.uk
Received September 7, 2005. Revised October 14, 2005. Accepted October 14, 2005.
In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants.
Correspondence may also be addressed to Yasuko Kamisugi. Tel: +44 113 3433097; Fax: +44 113 3433144; Email: bmbyk{at}leeds.ac.uk
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