Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of differentiation both in vitro and in vivo have been established. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide inexhaustible source of karyoplasts in nuclear transfer (NT). In this study the efficiencies of NT using porcine EG and fetal fibroblast cells were compared. Two different techniques were used to perform NT. With conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes, the rates of development to the blastocyst stage in EG and somatic cell NT were 16.8% (59/351) and 14.5% (98/677), respectively. In piezo-driven microinjection (Honolulu method) of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 9.4% (9/96), respectively. Regardless of NT methods used in this study, EG cell NT gave rise to comparable rate of blastocyst development to somatic cell NT. Overall, EG cells can be used as karyoplast donor in NT procedure, and embryos can be produced by EG cell NT that may be used as an alternative to conventional somatic cell NT.