Derivation and characterization of pluripotent porcine cells

尽管在世界范围内进行了大量的研究，但在国内物种中尚未获得稳定的多能胚胎干细胞（ESC）系。虽然在猪胚胎生殖细胞（pEGC）的衍生方面取得了一些进展，但衍生的细胞不能形成种系嵌合体。在这里，我们将采用一种新的方法，通过多西环素调节外源多能性基因的表达，在培养中阻止内细胞群（ICM）和/或外胚层细胞的分化，从而产生生殖系能力强的猪ESC。此外，我们将利用诱导多能干细胞（iPSC）的最新进展，通过诱导转录因子0CT4、S0X2和NANOG的表达，改善pEGCs的重编程以完成多能性，并研究表观遗传重编程对嵌合体生产效率的影响。两种来源的多能性细胞将以形态学、体外培养生长、多能性基因的表达、分化潜力和致瘤潜力为特征。我们期望这项研究能够建立用于生殖系嵌合体生产的猪多能细胞系，从而成为提高转基因猪生产效率的新工具，对人类和动物的健康产生重大影响。

Despite considerable world wide research efforts stable pluripotent embryonicstem cell (ESC) lines could not yet be derived in the domestic species. While someprogress has been achieved with the derivation of porcine embryonic germ cells (pEGC),derived cells were not able to form germ line chimeras. Here, we will use a novelapproach to produce germ line competent porcine ESC by preventing the differentiationof inner cell mass (ICM) and/or epiblast cells in culture by doxycycltne regulatedexpression of exogenous pluripotency genes. Additionally, we will use the recentprogress in the generation of induced pluripotent stem cells (iPSC) to improvereprogramming of pEGCs to complete pluripotency via induced expression of thetranscription factors 0CT4, S0X2, and NANOG, and investigate the effect of epigeneticreprogramming on the efficiency of chimera production. Putative pluripotent cells fromboth sources will be characterized by morphology, in vitro culture growth, expressionprofile of pluripotency genes, differentiation potential, and tumorigenic potential. Weanticipate that this study will result in the establishment of porcine pluripotent cell linesthat can be used for the production of germ line chimeras, and will thus constitute anovel tool for improving the efficiency of transgenic pig production with great impact onhuman and animal health.