Analysis of microRNAs in a knock-in hESC line expressing epitope-tagged AGO2

ABSTRACTArgonaute (AGO) family proteins, which are key components of RNA-induced silencing complexes (RISCs), associate with both microRNAs (miRNAs) and mRNAs to regulate gene expression at post-transcriptional stages. While high-quality antibodies for AGO proteins are extremely useful for studies on the functions of miRNAs and AGO proteins, they are not always readily available. In this study, we generated a knock-in human embryonic stem cell (hESC) line by using homologous recombination to express FLAG-tagged AGO2 from its native genomic locus. FLAG-tagged AGO2 was exploited to analyze AGO2-bound miRNAs in hESCs cultured on a feeder layer by immunoprecipitation. Furthermore, the pluripotency of ESCs allowed for the analysis of miRNAs after differentiation into fibroblasts and cardiomyocytes. The genetically modified hESC line generated in this study will be highly useful for studying the functions of miRNA-mediated regulation in human development and cell differentiation.