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

点击翻译按钮获取中文摘要

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.