This study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4, which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in fibroblast growth factor (FGF)- or leukemia inhibitory factor (LIF)-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. Porcine embryonic fibroblasts were coinfected with the pOCT4-ΔPE-eGFP (distal enhancer [DE]-green fluorescent protein [GFP]) and pOCT4-ΔDE-DsRed2 (proximal enhancer [PE]-red fluorescent protein [RFP]) vectors, and GFP and RFP expression were verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 DE and PE were activated in different expression patterns simultaneously as changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems confirmed that porcine iPSCs are in primed state after reprogramming in FGF2- or LIF-containing media. This work demonstrates the applicability of porcine OCT4 upstream region-derived dual fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.

中文翻译：

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猪 OCT4 报告系统可以在体细胞重编程过程中监测物种特异性多能性

本研究检测了猪OCT4增强剂在猪重编程细胞中的活性和功能。由OCT4上游调控区控制的双荧光蛋白报告系统是多能性的主要调控因子之一，广泛应用于多能性机制的研究。我们使用先前建立的猪特异性报告系统分析了该报告系统如何在成纤维细胞生长因子 (FGF) 或白血病抑制因子 (LIF) 依赖的重编程猪多能干细胞中发挥作用。猪胚胎成纤维细胞与 p OCT4 -ΔPE-eGFP（远端增强子 [DE]-绿色荧光蛋白 [GFP]）和 p OCT4共感染-ΔDE-DsRed2（近端增强子 [PE]-红色荧光蛋白 [RFP]）载体以及 GFP 和 RFP 表达在 DOX 依赖性重编程过程中得到验证。我们证明了猪OCT4 DE 和 PE 在建立猪诱导的多能干细胞 (iPSC) 期间随着多能标记基因表达的变化同时以不同的表达模式被激活。猪OCT4上游区域衍生的双荧光蛋白报告系统证实，猪 iPSC 在含有 FGF2 或 LIF 的培养基中重新编程后处于启动状态。这项工作证明了猪OCT4的适用性上游区域衍生的双荧光报告系统，可应用于猪源细胞中物种特异性多能性的研究。这些报告系统可能是研究猪特异性多能性、早期胚胎发育和胚胎干细胞的有用工具。