High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene

Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12265-9. doi: 10.1073/pnas.0502601102. Epub 2005 Aug 10.

Abstract

Gene targeting, which is homologous recombination-mediated integration of an extra-chromosomal DNA segment into a chromosomal target sequence, enables the precise disruption or replacement of any gene. Despite its value as a molecular genetic tool, gene targeting remains an inefficient technology in most species. We report that expression of the yeast RAD54 gene, a member of the SWI2/SNF2 chromatin remodeling gene family, enhances gene targeting in Arabidopsis by one to two orders of magnitude, from 10(-4) to 10(-3) in WT plants to 10(-2) to 10(-1). We show that integration events, detected with an assay based on the use of a fluorescent seed marker, are precise and germinally transmitted. These findings suggest that chromatin remodeling is rate-limiting for gene targeting in plants and improves the prospects for using gene targeting for the precise modification of plant genomes.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens
  • Arabidopsis / genetics*
  • Blotting, Southern
  • Chromatin Assembly and Disassembly / genetics*
  • DNA Helicases
  • DNA Primers
  • DNA Repair Enzymes
  • Gene Targeting / methods*
  • Green Fluorescent Proteins
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • DNA Primers
  • Saccharomyces cerevisiae Proteins
  • Green Fluorescent Proteins
  • RAD54 protein, S cerevisiae
  • DNA Helicases
  • DNA Repair Enzymes