TY - JOUR
T1 - Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines
AU - Donai, Kenichiro
AU - Kuroda, Kengo
AU - Guo, Yijie
AU - So, Kyoung Ha
AU - Sone, Hideko
AU - Kobayashi, Masayuki
AU - Nishimori, Katsuhiko
AU - Fukuda, Tomokazu
PY - 2013/1/1
Y1 - 2013/1/1
N2 - 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.
AB - 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.
KW - Gene silencing
KW - Induced pluripotent stem cells
KW - Reporter system
KW - Reprogramming
UR - http://www.scopus.com/inward/record.url?scp=84884918495&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84884918495&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2013.08.014
DO - 10.1016/j.ab.2013.08.014
M3 - Article
C2 - 23973628
AN - SCOPUS:84884918495
VL - 443
SP - 104
EP - 112
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -