title: A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA.
abstract: Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin resistance and the diphtheria toxin A subunit gene as positive and negative selection markers, respectively, is now available. However, to date, this selection system has been applied exclusively in rice (Oryza sativa). To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of neomycin phosphotransferaseII (nptII) and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic geneticin. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knockout of the endogenous rice genes Waxy and 33-kD globulin through GT, although negative selection with this system is relatively less efficient compared with diphtheria toxin A subunit. The approach developed here, with some additional improvements, could be applied as a universal selection system for the enrichment of GT cells in several plant species.
authors.affiliation: Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan (A.N.-Y., S.N., K.O., H.S., S.T.); Center for Collaboration among Agriculture, Industry and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); and Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Ayako
authors.identifier: http://orcid.org/0000-0003-2781-1359
authors.initials: A
authors.lastname: Nishizawa-Yokoi
authors.affiliation: Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan (A.N.-Y., S.N., K.O., H.S., S.T.); Center for Collaboration among Agriculture, Industry and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); and Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Satoko
authors.initials: S
authors.lastname: Nonaka
authors.affiliation: Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan (A.N.-Y., S.N., K.O., H.S., S.T.); Center for Collaboration among Agriculture, Industry and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); and Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Keishi
authors.initials: K
authors.lastname: Osakabe
authors.affiliation: Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan (A.N.-Y., S.N., K.O., H.S., S.T.); Center for Collaboration among Agriculture, Industry and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); and Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Saika
authors.affiliation: Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan (A.N.-Y., S.N., K.O., H.S., S.T.); Center for Collaboration among Agriculture, Industry and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); and Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.) stoki@affrc.go.jp.
authors.forename: Seiichi
authors.identifier: http://orcid.org/0000-0003-0066-6446
authors.initials: S
authors.lastname: Toki
mesh_terms: D000483:Alleles; D015139:Blotting, Southern; D020090:Chromosome Segregation / Q000187:drug effects; D004352:Drug Resistance, Microbial / Q000187:drug effects / Q000235:genetics*; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D017343:Genes, Plant; D056426:Genetic Loci; D005839:Gentamicins / Q000494:pharmacology; D012275:Oryza / Q000235:genetics; D010641:Phenotype; D030821:Plants, Genetically Modified; D016372:RNA, Antisense / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D005839:Gentamicins; D016372:RNA, Antisense; D012333:RNA, Messenger; C010680:antibiotic G 418