Usefulness of a non-invasive reporter system for monitoring reprogramming state in pig cells: results of a cell fusion experiment

Akio Ozawa, Eri Akasaka, Satoshi Watanabe, Mitsutoshi Yoshida, Kazuchika Miyoshi, Masahiro Sato
Journal of Reproduction and Development 2010, 56 (4): 363-9
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.

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