Usefulness of a non-invasive reporter system for monitoring reprogramming state in pig cells: results of a cell fusion experiment.
Dedifferentiation of differentiated cells such as fibroblasts into pluripotent stem cells, so-called iPS cells, was first reported by Yamanaka et al., who successfully employed retroviral gene delivery of four stem-cell-specific transcription factors (Oct-3/4, Klf4, Sox2 and c-myc). Despite the mouse system in which an Oct-3/4 or Nanog promoter-based reporter system has already been established, there is no useful system in pigs for reporting the reprogramming state of gene-engineered cells. In this study, we constructed a pOEIN plasmid carrying a ca. 5.4-kb mouse Oct-3/4 promoter linked to the EGFP cDNA and neomycin expression unit and produced a porcine embryonic cell line stably incorporating it in the genome. Cell fusion with mouse embryonal carcinoma cell line F9 resulted in generation of colonies with distinct EGFP-derived fluorescence around 14 days after fusion. RT-PCR using these colonies also confirmed expression of endogenous porcine pluripotency-specific Oct-3/4, Sox2 and Stat3 mRNA. These findings suggest that mouse-derived components are sufficient to induce dedifferentiation of differentiated pig cells and also that reprogramming proceeds gradually. The present non-invasive reporter system will be useful to better define the reprogramming mechanism and/or to identify novel reprogramming molecules in the pig.
Ozawa
Akio
A
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
Akasaka
Eri
E
Watanabe
Satoshi
S
Yoshida
Mitsutoshi
M
Miyoshi
Kazuchika
K
Sato
Masahiro
M
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002459:Cell Fusion / Q000379:methods*; D002460:Cell Line; D065150:Cellular Reprogramming / Q000235:genetics / Q000502:physiology*; D004526:Efficiency; D017930:Genes, Reporter / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D041681:NIH 3T3 Cells; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014162:Transfection
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins