Abstract
MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the post-transcriptional level. Different types of cells express unique sets of miRNAs that can be exploited as potential molecular markers to identify specific cell types. Among the variety of miRNA detection methods, a fluorescence-based imaging system that utilises a fluorescent-reporter gene regulated by a target miRNA offers a major advantage for long-term tracking of the miRNA in living cells. In this study, we developed a novel fluorescence-based miRNA-monitoring system using a non-integrating cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because SeVdp vectors robustly and stably express transgenes, this system enabled sensitive monitoring of miRNAs by fluorescence microscopy. By applying this system for cellular reprogramming, we found that miR-124, but not miR-9, was significantly upregulated during direct neuronal conversion. Additionally, we were able to isolate integration-free human induced pluripotent stem cells by long-term tracking of let-7 expression. Notably, this system was easily expandable to allow detection of multiple miRNAs separately and simultaneously. Our findings provide insight into a powerful tool for evaluating miRNA expression during the cellular reprogramming process and for isolating reprogrammed cells potentially useful for medical applications.
Highlights
MicroRNAs are a class of small noncoding RNAs that act as post-transcriptional regulators of gene expression[1]
To design an ideal fluorescence-based imaging system, we considered an optimal vector platform that continuously expresses a reporter gene regardless of cell type
Because the SeVdp vector is capable of stably expressing transgenes in various mammalian cells, we initially ascertained whether the SeVdp vector could maintain robust gene expression during in vitro differentiation of human induced pluripotent stem cells (hiPSCs)
Summary
MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as post-transcriptional regulators of gene expression[1]. The activities of cellular and viral promoters, which are commonly used to drive reporter gene expression, vary considerably depending on cell type, genomic context, and epigenetic control[17,18,19,20] Such characteristics might impede sensitive and stable monitoring of miRNAs during cell differentiation and reprogramming processes. These systems have been successfully applied for isolation of hiPSCs after somatic cell reprogramming[21,22], chromosomal integration of reporter genes is a critical disadvantage for the safe use of hiPSC-derived cells for clinical applications To overcome these limitations, we designed a novel fluorescence-based miRNA-monitoring system using a replication-defective and persistent Sendai virus (SeVdp) vector. These unique properties make the SeVdp vector a versatile gene delivery tool for various applications[27]
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