Establishing pig embryonic stem cells (pESCs) remains a challenge due to differences in the genetic backgrounds of mouse, human, and pig. Therefore, pig-specific pluripotency markers and cellular signaling must be identified. In this study, doxycycline (DOX)-inducible vectors carrying Oct4, sex-determining region Y-box 2 (Sox2), Nanog, Kruppel-like family 4 (Klf4), or Myc, which are known reprogramming factors, were transduced into pESCs. And pluripotency genes were analyzed in one or two reprogramming factor-expressed pESCs. When cultured without DOX, pESCs were stably maintained in basic fibroblast growth factor-supplemented media. However, when treated with DOX, the cells lost their alkaline phosphatase (AP) activity and differentiated within 2 weeks. Subsequently, we investigated the expression of genes related to pluripotency in DOX-treated pESCs using quantitative reverse transcription-polymerase chain reaction (PCR). Expression levels of Oct4, E-cadherin, and Fut4 were significantly increased by Oct4 overexpression, and Oct4 and Fut4 were upregulated in the Sox2-infected group. When a combination of two reprogramming factors, including Oct4 or Sox2, was introduced, weak AP activity remained. In addition, several of the two reprogramming factor transduction groups could be maintained after subculturing with transgene activation. Although long-term culture failed, pESCs transduced with Oct4 and Nanog, Oct4 and Klf4, or Sox2 and Nanog combinations could be subcultured even under transgene activation conditions. Analysis of the cause of long-term culture failure by quantitative PCR confirmed that the expression of intermediate reprogramming markers was not maintained. Given these results, additional methods are needed to support the completion of each reprogramming phase to succeed in the conversion of the pluripotent state of pESCs. This study improves our understanding of pluripotent networks and can be used to aid in the establishment of bona fide pig pluripotent stem cells.

中文翻译：

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尝试通过重编程因子的过表达将致敏的猪胚胎干细胞转变为幼稚状态。

由于小鼠，人和猪的遗传背景不同，建立猪胚胎干细胞（pESCs）仍然是一个挑战。因此，必须识别猪特异性多能性标志物和细胞信号传导。在这项研究中，将已知的重编程因子携带Oct4，性别决定区域Y-box 2（Sox2），Nanog，Kruppel样家族4（Klf4）或Myc的强力霉素（DOX）诱导型载体转导入pESCs。 。并在一个或两个重编程因子表达的pESC中分析了多能性基因。当不添加DOX进行培养时，pESC可以稳定地补充在碱性成纤维细胞生长因子补充培养基中。但是，当用DOX处理时，细胞会失去其碱性磷酸酶（AP）活性并在2周内分化。后来，我们使用定量逆转录-聚合酶链反应（PCR）研究了在DOX处理的pESC中与多能性相关的基因的表达。在Sox2感染组，Oct4，E-cadherin和Fut4的表达水平显着增加，并且Oct4和Fut4被上调。当引入两个重编程因子（包括Oct4或Sox2）的组合时，AP活性仍然很弱。另外，在用转基因激活继代培养后，可以维持两个重编程因子转导组中的几个。尽管长期培养失败，但即使在转基因激活条件下，用Oct4和Nanog，Oct4和Klf4或Sox2和Nanog组合转导的pESC仍可以继代培养。通过定量PCR分析长期培养失败的原因证实，中间重编程标记的表达没有得到维持。鉴于这些结果，需要其他方法来支持每个重新编程阶段的完成，以成功完成pESC的多能状态转换。这项研究提高了我们对多能网络的理解，可用于帮助建立真正的猪多能干细胞。