| RRC ID |
54548
|
| Author |
Donai K, Kuroda K, Guo Y, So KH, Sone H, Kobayashi M, Nishimori K, Fukuda T.
|
| Title |
Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines.
|
| Journal |
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.
|
| Volume |
443(1)
|
| Pages |
104-12
|
| Published |
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
|
| Times Cited |
14
|
| Resource |
| DNA material |
pCMV-VSV-G-RSV-Rev (RDB04393)
pCAG-HIVgp (RDB04394) |
| Human and Animal Cells |
201B7(HPS0063) |
