Real-time functional imaging for monitoring miR-133 during myogenic differentiation |
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Authors: | Yoshio Kato Shigeru Miyaki Shigetoshi Yokoyama Shin Omori Atsushi Inoue Machiko Horiuchi Hiroshi Asahara |
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Institution: | aResearch Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba 305-8562, Japan;bNational Institute for Child Health and Development, Tokyo, Japan;cThe Scripps Research Institute, La Jolla, CA, USA |
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Abstract: | MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events. |
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Keywords: | Myogenesis miR-133 Retroviral vector Fluorescent protein Live-cell imaging |
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