title: Long time-course monitoring of ZFP809-mediated gene silencing in transgene expression driven by promoters containing MLV-derived PBS.
abstract: Expression of Moloney murine leukemia virus (MoMLV)-typed retroviral vectors is strictly suppressed in immature cells such as embryonic stem cells. The phenomenon known as gene silencing is primed by the sequence-specific binding of the zinc finger protein 809 (ZFP809) to the primer-binding site of the vectors. However, it has yet to be determined whether the ZFP809-mediated gene silencing is maintained over a long period. In this study, we established an experimental system that can monitor gene silencing during a long-term cell culture using flow cytometry technology combined with fluorescent reporters for the expression of ZFP809 and the transgene expression driven by the promoters of interest. Time-course analysis using our system revealed that ZFP809 maintains gene silencing effect even at a longtime period. Furthermore, our system was useful for the monitoring of ZFP809-mediated gene silencing regardless of the types of vectors and cell lines.
authors.affiliation: a Department of Human Genetics , National Research Institute for Child Health and Development , Tokyo , Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Ichida
authors.affiliation: a Department of Human Genetics , National Research Institute for Child Health and Development , Tokyo , Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Utsunomiya
authors.affiliation: b Department of Maternal-Fetal Biology , National Research Institute for Child Health and Development , Tokyo , Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Tomikawa
authors.affiliation: b Department of Maternal-Fetal Biology , National Research Institute for Child Health and Development , Tokyo , Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Nakabayashi
authors.affiliation: c Department of Biosciences and Informatics , Keio University , Yokohama , Japan.
authors.forename: Toshinori
authors.initials: T
authors.lastname: Sato
authors.affiliation: a Department of Human Genetics , National Research Institute for Child Health and Development , Tokyo , Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Onodera
mesh_terms: D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008979:Moloney murine leukemia virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013234:Stem Cells / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors
chemical_list: D004268:DNA-Binding Proteins; C541321:ZFP809 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: gene silencing; retroviral vector; zinc finger protein 809 (ZFP809)