title: Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line, Pv11.
abstract: Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5-34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.
authors.affiliation: Anhydrobiosis Research Group, Molecular Biomimetics Research Unit, Institute of Agrobiological Sciences, NARO (NIAS), Tsukuba, Japan.|Japan Society for the Promotion of Science, Tokyo, Japan.
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Sogame
authors.affiliation: Anhydrobiosis Research Group, Molecular Biomimetics Research Unit, Institute of Agrobiological Sciences, NARO (NIAS), Tsukuba, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Okada
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kikuta
authors.affiliation: Anhydrobiosis Research Group, Molecular Biomimetics Research Unit, Institute of Agrobiological Sciences, NARO (NIAS), Tsukuba, Japan.|Center for Biological Resources and Informatics, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan.
authors.forename: Yugo
authors.initials: Y
authors.lastname: Miyata
authors.affiliation: Anhydrobiosis Research Group, Molecular Biomimetics Research Unit, Institute of Agrobiological Sciences, NARO (NIAS), Tsukuba, Japan.
authors.forename: Richard
authors.initials: R
authors.lastname: Cornette
authors.affiliation: Institute of Fundamental Biology and Medicine, Kazan Federal University, Kazan, Russia.|Division of Genomic Technologies, Center for Life Science Technologies, RIKEN, Yokohama, Japan.|Preventive Medicine and Diagnosis Innovation Program, Center for Life Science Technologies, RIKEN, Yokohama, Japan.
authors.forename: Oleg
authors.initials: O
authors.lastname: Gusev
authors.affiliation: Anhydrobiosis Research Group, Molecular Biomimetics Research Unit, Institute of Agrobiological Sciences, NARO (NIAS), Tsukuba, Japan. kikawada@affrc.go.jp.|Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan. kikawada@affrc.go.jp.
authors.forename: Takahiro
authors.identifier: http://orcid.org/0000-0002-2329-9282
authors.initials: T
authors.lastname: Kikawada
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002683:Chironomidae / Q000166:cytology / Q000235:genetics*; D003890:Desiccation; D018014:Gene Transfer Techniques; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D019476:Insect Proteins / Q000235:genetics; D007814:Larva / Q000166:cytology; D034622:RNA Interference; D013312:Stress, Physiological / Q000235:genetics
chemical_list: D019476:Insect Proteins; D049452:Green Fluorescent Proteins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
keywords: Anhydrobiosis; Desiccation tolerant cells; Gene expression system; RNAi