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

全基因组鉴定细胞分化的非编码 RNA 功能

• 批准号: 13F03717
• 负责人: CARNINCI Piero
• 金额: $ 1.47万
• 依托单位: The Institute of Physical and Chemical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2013
• 资助国家: 日本
• 起止时间: 2013 至 2014
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13F03717/
• 关键词: ncRNA; mESC; pluripotency; mES; iPS

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.

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用于将MESC保持在未分化状态。在用编码NCRNA的表达向量转染细胞后几个小时，我们从MESC中撤出了LIF。如果NCRNA不参与腐烂的维持，则MESC将自发区分；相反，如果NCRNA与保持细胞未分化有关，则转染后MESC将保持多能状态。 48小时后纳米表达水平的检测将用于评估MESC是否保留其未分化的表型。相应地，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纳米水平。我们从此类转染中收集了总RNA，并产生了笼子库以及正面和阴性对照。 results提出了一个数量或蛋白质编码基因，这些基因在过表达后被下调，并且在转染后受差异调节的许多转录本。产生的数据表明，涉及MESC中多能维持的基因网络。