RRC ID 54548
著者 Donai K, Kuroda K, Guo Y, So KH, Sone H, Kobayashi M, Nishimori K, Fukuda T.
タイトル Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines.
ジャーナル Anal Biochem
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.
巻・号 443(1)
ページ 104-12
公開日 2013-12-1
DOI 10.1016/j.ab.2013.08.014
PII S0003-2697(13)00398-9
PMID 23973628
MeSH Animals Cell Differentiation Cellular Reprogramming / genetics Embryo, Mammalian Fibroblasts / cytology Fibroblasts / metabolism Gene Silencing* Genes, Reporter* Genetic Engineering / methods* Genetic Vectors Green Fluorescent Proteins / antagonists & inhibitors Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism HEK293 Cells Humans Induced Pluripotent Stem Cells / cytology Induced Pluripotent Stem Cells / metabolism* Lentivirus / genetics* Luciferases / antagonists & inhibitors Luciferases / genetics Luciferases / metabolism Mice Transgenes
IF 2.877
引用数 14
リソース情報
遺伝子材料 pCMV-VSV-G-RSV-Rev (RDB04393) pCAG-HIVgp (RDB04394)
ヒト・動物細胞 201B7(HPS0063)