title: Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red AlgaCyanidioschyzon merolae.
abstract: The unicellular red algaCyanidioschyzon merolaepossesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection markerURAhas been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of theCATselection marker into the nuclear genome. By means of a combination of theURAandCATtransformation systems, we succeeded in producing aC. merolaestrain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomalCYC1andCDKAloci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation ofC. merolae.
authors.affiliation: Department of Cell Genetics, National Institute of GeneticsShizuoka, Japan; Japan Science and Technology Agency, Core Research for Evolutional Science and TechnologySaitama, Japan; Department of Genetics, Graduate University for Advanced StudiesShizuoka, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Fujiwara
authors.affiliation: Japan Science and Technology Agency, Core Research for Evolutional Science and TechnologySaitama, Japan; National Institute of Technology, Hiroshima CollegeHiroshima, Japan.
authors.forename: Mio
authors.initials: M
authors.lastname: Ohnuma
authors.affiliation: Japan Science and Technology Agency, Core Research for Evolutional Science and TechnologySaitama, Japan; Department of Chemical and Biological Science, Faculty of Science, Japan Women's UniversityTokyo, Japan.
authors.forename: Tsuneyoshi
authors.initials: T
authors.lastname: Kuroiwa
authors.affiliation: Department of Cell Genetics, National Institute of GeneticsShizuoka, Japan; Japan Science and Technology Agency, Core Research for Evolutional Science and TechnologySaitama, Japan.
authors.forename: Ryudo
authors.initials: R
authors.lastname: Ohbayashi
authors.affiliation: Department of Cell Genetics, National Institute of GeneticsShizuoka, Japan; Japan Science and Technology Agency, Core Research for Evolutional Science and TechnologySaitama, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Hirooka
authors.affiliation: Department of Cell Genetics, National Institute of GeneticsShizuoka, Japan; Japan Science and Technology Agency, Core Research for Evolutional Science and TechnologySaitama, Japan; Department of Genetics, Graduate University for Advanced StudiesShizuoka, Japan.
authors.forename: Shin-Ya
authors.initials: SY
authors.lastname: Miyagishima
keywords: CAT selection marker; algae; chloramphenicol; genetic modification; multiple transformation; photosynthetic eukaryotes; plants