細胞分化のためのnon-coding RNA機能のゲノム全体における識別

在我的JSPS奖学金的第二年，我已经能够建立一种筛选方法来评估非编码RNA（ncRNA）在维持多能性中的作用。在表达载体中克隆了大约100个在小鼠胚胎干细胞（mESC）中特异性表达的ncRNA后，我们开发了一种白血病抑制因子（LIF）剥夺试验来筛选哪些ncRNA对于维持mESC处于未分化状态是重要的。在用编码ncRNA的表达载体转染细胞后几小时，我们从mESC培养基中取出LIF。如果ncRNA不参与维持纯能性，那么mESC将自发分化;相反，如果ncRNA参与保持细胞未分化，那么转染后mESC将维持多能状态。48小时后nanog表达水平的检测将用于评估mESC是否保留其未分化表型。随后，我开始筛选上述ncRNA，我们发现六个转录本能够在过表达时增加nanog的水平。我们从这样的转染中收获总RNA，并与阳性和阴性对照一起产生CAGE文库。研究结果表明，一些蛋白质编码基因在过表达时下调，以及一些转录本在转染后差异调节。所产生的数据表明，基因网络参与维持mESC的多能性。

During the second year of my JSPS fellowship I have been able to establish a screening methodology to assess the role of non-coding RNAs (ncRNAs) in the maintenance of pluripotency.Briefly after cloning in an expression vector around 100 ncRNAs that are specifically expressed in mouse embryonic stem cells (mESCs), we developed a leukemia inhibitory factor (LIF) deprivation assay to screen which ncRNAs are central for maintaining mESCs in an undifferentiated state. We withdrew LIF from the mESCs medium few hours after transfecting the cells with expression vectors encoding for ncRNAs. If the ncRNAs are not involved in the maintenance of puripotency then mESCs will spontaneously differentiate ; if instead the ncRNAs are involved in keeping the cell undifferentiated then upon transfection mESCs will maintain a pluripotent state. Detection of nanog expression levels after 48 hours will be used to assess if mESCs retain their undifferentiated phenotype. Susequently I started to screen the abovementioned ncRNAs and we found six transcripts to be able to increase the levels of nanog upon overexpression. We harvested the total RNA from such transfections and we generated CAGE libraries together with positive and negative controls. Rresults have suggested a number or protein coding genes that are downregulated upon overexpression as well as a number of transcripts that are differentially regulated after transfection. The produced data suggest a gene network involved in the maintenance of pluripotency in mESCs.