title: Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines.
abstract: Induced pluripotent stem (iPS) cells have proven to be an effective technology in regenerative medicine; however, the low efficiency of reprogramming is a major obstacle to the successful generation of iPS cell lines. One of the most important characteristics of a high-quality iPS cell line is the inactivation of transgenes driven by a retrovirus-derived long terminal repeat promoter. In this study, we established a novel marker system containing three kinds of proteins: secreted-type luciferase (MetLuc), copepod Pontellina plumata green fluorescent protein (copGFP), and an antibiotic-resistant gene product (Neo(r)). The introduction of MetLuc-copGFP-Neo(r) in mouse embryonic fibroblasts (MEFs) allowed us to monitor the reporter expression changes as an indicator of the state of silencing during reprogramming. Transformation of iPS cells induced a remarkable reduction in reporter activity, indicating that the retroviral silencing was detected successfully. Our system enables us to monitor the silencing status of transgenes and to efficiently select iPS cell lines that can be used for further applications.
authors: Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan.
authors: Kenichiro
authors: K
authors: Donai
authors: Kengo
authors: K
authors: Kuroda
authors: Yijie
authors: Y
authors: Guo
authors: Kyoung-Ha
authors: KH
authors: So
authors: Hideko
authors: H
authors: Sone
authors: Masayuki
authors: M
authors: Kobayashi
authors: Katsuhiko
authors: K
authors: Nishimori
authors: Tomokazu
authors: T
authors: Fukuda
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D065150:Cellular Reprogramming / Q000235:genetics; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020868:Gene Silencing; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019076:Transgenes
chemical_list: D049452:Green Fluorescent Proteins; D008156:Luciferases
keywords: Gene silencing; Induced pluripotent stem cells; Reporter system; Reprogramming