Mouse iPSC generated with porcine reprogramming factors as a model for studying the effects of non-silenced heterologous transgenes on pluripotency.

Mouse somatic cells can be reprogrammed to pluripotency by the ectopic expression of four pluripotency transcription factors, Oct4, Sox2, cmyc, and Klf4. Usually, silencing of the exogenous reprogramming factors is considered to be essential for complete reprogramming and differentiation. In the vast majority of studies, murine pluripotency transcription factor sequences have been used for the reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSC). The effectiveness of xenogeneic transcription factors in miPSC generation has not yet been investigated in detail. Here, we evaluated transposon-based vectors with four porcine pluripotency factors for their ability to reprogram mouse fetal fibroblasts (MEFs) harboring an Oct4-EGFP reporter construct to pluripotency. Additionally, we examined the effects of the non-silenced heterologous transgenes on the expression levels of key endogenous pluripotency markers and the differentiation capacities of the miPSC. Within 8 days of transfection with porcine reprogramming transcription factors the MEFs acquired typical compact miPSC morphology and upregulated expression of endogenous Oct4 and other critical pluripotency genes. Consequently, the transgenes under the control of the TetO promoter became silenced, while the CAG-controlled constructs were expressed throughout the period of culture. Despite the continuous transgene expression, the CAG-miPSC showed normal morphology and were capable of differentiation into the three primary germ layers