Characterization of the proximal region of the goat NANOG promoter that is used for monitoring cell reprogramming and early embryo development

Nanog is a key transcription regulatory molecule that plays an important role in maintaining stem cell pluripotency. However, the molecular features and transcription regulation of the NANOG gene in domestic animals are not well investigated. In this study, the 751-base pairs (bp) fragment of the proximal region of the goat NANOG promoter (GNP), which has a 572-bp promoter sequence retaining multiple transcription binding sites and a 179-bp 5′ untranslated region of the goat NANOG gene, was cloned and characterized.The recombinant construct of pGNP-EGFP (enhanced green fluorescent protein) was solely activated in pluripotent cells and could be upregulated by the Oct4/Sox2 complex. The construct was stably transfected into goat fetal fibroblast (GFF) cells that were then used as the recipient cells to generate the induced pluripotent stem (iPS) cells. GNP-directed EGFP expression could be used to monitor the progression of cell reprogramming and the formation of iPS cells. The pGNP-EGFP construct was also delivered into goat oocytes cultured in vitro by microinjection. Interestingly, NANOG expression pattern in early stage goat embryos matured in vitro was asymmetrical. In two-cell embryos, the expression level of NANOG was uneven with one blastomere expressing EGFP and the next blastomere with no expression of EGFP. This was also observed in four-cell embryos. This asymmetrical expression may be due to the heterozygous expression of NANOG because of the quality of embryos and the culture environment. In conclusion, the GNP-EGFP reporter system represents a useful tool to monitor endogenous NANOG activation and for research with goat pluripotent stem cells.