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首页> 外文期刊>BMC Biotechnology >Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters
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Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters

机译：用激活内源性启动子的四个转录因子mRNA诱导山羊多能干细胞

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BackgroundTraditional approaches for generating goat pluripotent stem cells (iPSCs) suffer from complexity and low preparation efficiency. Therefore, we tried to derive goat iPSCs with a new method by transfecting exogenous Oct4, Sox2, Klf4 and c-Myc mRNAs into goat embryonic fibroblasts (GEFs), and explore the mechanisms regarding the transcription regulation of the reprogramming factors in goat iPSCs induction. ResultsmRNAs of the four reprogramming factors were transfected into GEFs, and were localized in nucleus with approximately 90% transfection efficiency. After five consecutive transfections, GEFs tended to aggregate by day 10. Clones appeared on day 15–18, and typical embryonic stem cell -like clones formed on day 20. One thousand AKP staining positive clones were achieved in 104 GEFs, with approximately 1.0% induction efficiency. Immunofluorescence staining and qRT-PCR detection of the ESCs markers confirmed the properties of the goat iPSCs. The achieved goat iPSCs could be cultured to 22nd passage, which showed normal karyotype. The goat iPSCs were able to differentiate into embryoid bodies with three germ layers. qRT-PCR and western blot showed activated endogenous pluripotent factors expression in the later phase of mRNA-induced goat iPSCs induction. Epigenetic analysis of the endogenous pluripotent gene Nanog revealed its demethylation status in derived goat iPSCs. Core promoter regions of the four reprogramming factors were determined. Transcription factor binding sites, including Elf-1, AP-2, SP1, C/EBP and MZF1, were identified to be functional in the core promoter regions of these reprogramming genes. Demethylation and deacetylation of the promoters enhanced their transcription activities. ConclusionsWe successfully generated goat iPSCs by transfection of Oct4, Sox2, Klf4 and c-Myc mRNAs into GEFs, which initiated the endogenous reprogramming network and altered the methylation status of pluripotent genes. Core promoter regions and functional transcription binding sites of the four reprogramming genes were identified. Epigenetic regulation was revealed to participate in mRNA induced iPSCs formation. Our study provides a safe and efficient approach for goat. iPSCs generation.
机译：背景技术用于产生山羊多能干细胞（iPSC）的传统方法遭受复杂性和低制备效率的困扰。因此，我们尝试通过将外源Oct4，Sox2，Klf4和c-Myc mRNA转染到山羊胚胎成纤维细胞（GEFs）中的新方法来衍生山羊iPSC，并探索有关山羊iPSCs诱导中重编程因子转录调控的机制。结果将四种重编程因子的mRNAs转染到GEFs中，并以约90％的转染效率定位在细胞核中。经过五次连续转染后，GEF倾向于在第10天聚集。在第15-18天出现克隆，在第20天形成典型的胚胎干细胞样克隆。在10 4 中获得了千个AKP染色阳性克隆。 sup> GEF，感应效率约为1.0％。 ESCs标记的免疫荧光染色和qRT-PCR检测证实了山羊iPSC的特性。获得的山羊iPSCs可以培养到第22代，显示出正常的核型。山羊iPSC能够分化为具有三个胚层的胚状体。 qRT-PCR和蛋白质印迹显示在mRNA诱导的山羊iPSCs诱导的后期，活化的内源性多能因子表达。对内源性多能基因Nanog的表观遗传分析显示，其在衍生山羊iPSC中的脱甲基状态。确定了四个重编程因子的核心启动子区域。转录因子结合位点，包括Elf-1，AP-2，SP1，C / EBP和MZF1，被确定在这些重编程基因的核心启动子区域中起作用。启动子的去甲基化和去乙酰化增强了它们的转录活性。结论我们成功地通过将Oct4，Sox2，Klf4和c-Myc mRNA转染到GEF中成功产生了山羊iPSC，从而启动了内源性重编程网络并改变了多能基因的甲基化状态。确定了四个重编程基因的核心启动子区域和功能性转录结合位点。表观遗传调控被揭示参与mRNA诱导的iPSC的形成。我们的研究为山羊提供了一种安全有效的方法。 iPSC生成。

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