[Detection of dynamic expression of microRNAs in vivo using a dual-fluorescence reporter system/miRNA Tracer in zebrafish].

microRNAs (miRNAs) are short noncoding RNAs that have been found in a wide variety of organisms and many have been shown to play essential roles by regulating the stability and translation of target messenger RNAs (mRNAs) in animals and plants. Temporal and spatial expression is critical for the regulatory function of miRNAs. To analyze the dynamic expression of particular miRNA in vivo, we constructed a dual-fluorescence reporter system based on Tol2 transposon, in which two reporter genes, enhanced green fluorescent protein (eGFP) and monomeric red fluorescent protein 1 (mRFP1), were driven by the heat shock promoter (hsp) from zebrafish hsp70 gene in an opposite orientation. To sense the existence of a particular miRNA, the complementary DNA sequence of the corresponding miRNA was inserted into the 3'-UTR region of one of the two reporter genes. By injecting the corresponding plasmid DNA into zebrafish embryos, we were able to monitor the abundance and dynamics of miRNA miR-206 in live embryos. To further evaluate this method, we made a collection of transgenic zebrafish with stable integration of dual-fluorescence reporter plasmids targeting different miRNAs, including miR-206 and miR-219. Our results showed that this dual-fluorescence reporter system, which is also called miRNA Tracer, could faithfully monitor the appearance and disappearance of target miRNAs in defined cell lineages during zebrafish development in these fish lines. Our dual-fluorescence reporter/Tracer system provides an important tool for further in-depth studies on miRNAs in zebrafish.