Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines

Kenichiro Donai, Kengo Kuroda, Yijie Guo, Kyoung Ha So, Hideko Sone, Masayuki Kobayashi, Katsuhiko Nishimori, Tomokazu Fukuda

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

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 (Neor). The introduction of MetLuc-copGFP-Neor 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.

Original languageEnglish
Pages (from-to)104-112
Number of pages9
JournalAnalytical Biochemistry
Volume443
Issue number1
DOIs
Publication statusPublished - 2013 Jan 1

Keywords

  • Gene silencing
  • Induced pluripotent stem cells
  • Reporter system
  • Reprogramming

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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