Beneficial Effects of Reversine on In Vitro Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos

摘要

Reversine, a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog, can induce dedifferentiation of myogenic lineage-committed cells into multipotent mesenchymal progenitor cells, from which osteoblasts and adipocytes redifferentiate under lineage-specific inducing conditions. Although the molecular mechanism of how reversine causes dedifferentiation of a differentiated cell has not been fully elucidated, we speculated that it would be involved in reprogramming. In the present study, we examined whether reversine can enhance the development of somatic cell nuclear transfer (SCNT) embryos by improving the reprogramming state of the somatic cell nuclei. As donor cells, we used miniature pig fetal fibroblasts transfected with a plasmid construct containing a mouse Oct-3/4 promoter and enhanced green fluorescent protein (EGFP) cDNA. When the nuclei of these transfected cells are reprogrammed to an undifferentiated state in the SCNT embryos, EGFP expression is expected to commence under the control of the Oct-3/4 promoter. After SCNT, the resulting embryos were treated with 5 mu M reversine for different durations (0, 6, 12, 18 and 24 h) or at different concentrations (0, 1, 5 and 10 mu M) of reversine for 12 h and then cultured in vitro. When embryos were treated with 5 mu M reversine for 12 h, the blastocyst formation rate was significantly (P<0.01) higher than that of embryos without reversine treatment. However, the strength and pattern of EGFP expression in the embryos were not affected by the same treatment. A normal-looking fetus was obtained 21 days after transfer of embryos treated with 5 mu M reversine for 12 h into recipients. The present findings indicate that treatment with reversine is beneficial for enhancement of the in vitro development of miniature pig SCNT embryos, although the underlying mechanism is still unclear.