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Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination

  • Ayako Nishizawa-Yokoi
  • Hiroaki Saika
  • Seiichi TokiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1469)

Abstract

Positive–negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive–negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice.

Key word

Gene targeting Positive–negative selection Marker excision piggyBac transposon Homologous recombination 

Notes

Acknowledgments

We acknowledge Dr. R. Terada (Meijo University) and Dr. S. Iida (Shizuoka Pref. University) for providing the DT-A gene, Dr. K. Uchino and Dr. H. Sezutsu (National Institute of Agrobiological Sciences) for providing hyPBase gene, and Dr. H. Rothnie for English editing. This research was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation PGE1001), KAKENHI (23658012 and 23310142) and the Cross-ministerial Strategic Innovation Promotion Program (SIP).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ayako Nishizawa-Yokoi
    • 1
  • Hiroaki Saika
    • 1
  • Seiichi Toki
    • 1
    • 2
    Email author
  1. 1.Plant Genome Engineering Research UnitNational Institute of Agrobiological SciencesTsukubaJapan
  2. 2.Kihara Institute for Biological ResearchYokohama City UniversityYokohamaJapan

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