High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene.
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.
Shaked
Hezi
H
Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel.
Melamed-Bessudo
Cathy
C
Levy
Avraham A
AA
D016960:Agrobacterium tumefaciens; D017360:Arabidopsis / Q000235:genetics*; D015139:Blotting, Southern; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D004265:DNA Helicases; D017931:DNA Primers; D045643:DNA Repair Enzymes; D018390:Gene Targeting / Q000379:methods*; D049452:Green Fluorescent Proteins; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes