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 develop-ment in these fish lines. Our dual-fluorescence reporter/Tracer system provides an important tool for further in-depth studies on miRNAs in zebrafish.
[1] Pasquinelli AE. MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet, 2012, 13(4): 271-282.
[2] 张磊, 晁江涛, 崔萌萌, 陈雅琼, 宗鹏, 孙玉合. 茄子microRNAs与其靶基因的生物信息学预测. 遗传, 2011, 33(7): 776-784.
[3] 刘利英, 徐纪茹, 宋土生, 黄辰. miRNA及其靶位点多态性的研究进展. 遗传, 2010, 32(11): 1091- 1096.
[4] Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell, 2012, 148(6): 1172-1187.
[5] 景花, 宋沁馨, 周国华. MicroRNA定量检测方法的研究进展. 遗传, 2010, 32(1): 31-40.
[6] Koscianska E, Starega-Roslan J, Czubala K, Krzyzosiak WJ. High-resolution northern blot for a reliable analysis of microRNAs and their precursors. Sci World J, 2011, 11: 102-117.
[7] D'Andrade PN, Fulmer-Smentek S. Agilent microRNA microarray profiling system. Methods Mol Biol, 2012, 822: 85-102.
[8] 汤海明, 陈红, 张静, 任景怡, 许宁. 新一代测序技术应用于microRNA检测. 遗传, 2012, 34(6): 784 -792.
[9] Wu Q, Lu ZH, Li HL, Lu JF, Guo L, Ge QY. Next-generation sequencing of microRNAs for breast cancer detection. J Biomed Biotechnol, 2011, 2011: 597145.
[10] Benes V, Castoldi M. Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods, 2010, 50(4): 244-249.
[11] Chen CF, Tan RY, Wong LD, Fekete R, Halsey J. Quantitation of microRNAs by real-time RT- qPCR. Methods Mol Biol, 2011, 687: 113-134.
[12] Tang FC, Hajkova P, Barton SC, Lao KQ, Surani MA. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res, 2006, 34(2): e9.
[13] Silahtaroglu AN, Nolting D, Dyrskjot L, Berezikov E, Møller M, Tommerup N, Kauppinen S. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. Nat Protoc, 2007, 2(10): 2520-2528.
[14] Várallyay E, Burgyán J, Havelda Z. MicroRNA detection by northern blotting using locked nucleic acid probes. Nat Protoc, 2008, 3(2): 190-196.
[15] Mansfield JH, Harfe BD, Nissen R, Obenauer J, Srineel J, Chaudhuri A, Farzan-Kashani R, Zuker M, Pasquinelli AE, Ruvkun G, Sharp PA, Tabin CJ, McManus MT. MicroRNA- responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat Genet, 2004, 36(10): 1079-1083.
[16] Johnson SM, Lin SY, Slack FJ. The time of appearance of the C. elegans let-7 microRNA is transcriptionally controlled utilizing a temporal regulatory element in its promoter. Dev Biol, 2003, 259(2): 364-379.
[17] De Pietri Tonelli D, Calegari F, Fei JF, Nomura T, Osumi N, Heisenberg CP, Huttner WB. Single-cell detection of microRNAs in developing vertebrate embryos after acute administration of a dual-fluorescence reporter/sensor plasmid. Biotechniques, 2006, 41(6): 727-732.
[18] 丁雷, 闫学春, 孙效文, 滕春波. MicroRNAs对斑马鱼发育的调控. 遗传, 2011, 33(11): 1179- 1184.
[19] Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, de Bruijn E, Horvitz HR, Kauppinen S, Plasterk RHA. MicroRNA expression in zebrafish embryonic development. Science, 2005, 309(5732): 310-311.
[20] Wienholds E, Plasterk RH. MicroRNA function in animal development. FEBS Lett, 2005, 579(26): 5911-5922.
[21] Rao PK, Kumar RM, Farkhondeh M, Baskerville S, Lodish HF. Myogenic factors that regulate expression of muscle-specific microRNAs. Proc Natl Acad Sci USA, 2006, 103(23): 8721- 8726.
[22] Fu YS, Shi ZY, Wang GY, Li WJ, Zhang JL, Jia L. Expression and regulation of miR-1, -133a, -206a, and MRFs by thyroid hormone during larval development in Paralichthys olivaceus. Comp Biochem Physiol B Biochem Mol Biol, 2012, 161(3): 226-232.
[23] Shkumatava A, Stark A, Sive H, Bartel DP. Coherent but overlapping expression of microRNAs and their targets during vertebrate development. Genes Dev, 2009, 23(4): 466-481
