title: Precise marker excision system using an animal-derived piggyBac transposon in plants.
abstract: Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth-derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase-expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T0 plants without concomitant re-integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.
authors.affiliation: Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Nishizawa-Yokoi
authors.forename: Masaki
authors.initials: M
authors.lastname: Endo
authors.forename: Keishi
authors.initials: K
authors.lastname: Osakabe
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Saika
authors.forename: Seiichi
authors.initials: S
authors.lastname: Toki
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics*; D018745:Genome, Plant / Q000235:genetics*; D009036:Moths; D009154:Mutation; D012275:Oryza / Q000235:genetics*; D019076:Transgenes
chemical_list: D004251:DNA Transposable Elements; D005819:Genetic Markers
keywords: Marker excision; marker-free plant; piggyBac; precision genome engineering; rice; technical advance; transposon