应用双荧光报告系统检测斑马鱼microRNA的动态表达

doi:10.3724/SP.J.1005.2012.01181

遗传 ›› 2012, Vol. 34 ›› Issue (9): 1181-1192.doi: 10.3724/SP.J.1005.2012.01181

应用双荧光报告系统检测斑马鱼microRNA的动态表达

杨红波¹, 梁巍¹, 刘新星¹, 朱作言¹, 林硕², 张博¹

1. 北京大学生命科学学院, 细胞增殖与分化教育部重点实验室, 北京 100871 2. 美国加州大学洛杉矶分校, 分子、细胞与发育生物学系, 洛杉矶 90095-1606

收稿日期:2012-06-18 修回日期:2012-07-16 出版日期:2012-09-20 发布日期:2012-09-25
通讯作者: 张博 E-mail:bzhang@pku.edu.cn
基金资助:
国家重大科学研究计划项目(编号：2012CB945101)和国家自然科学基金项目(编号：31110103904)资助

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

YANG Hong-Bo¹, LIANG Wei¹, LIU Xin-Xing¹, ZHU Zuo-Yan¹, LIN Shuo², ZHANG Bo¹

1. Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Bei-jing 100871, China 2. Department of Molecular, Cell Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095-1606, USA

Received:2012-06-18 Revised:2012-07-16 Online:2012-09-20 Published:2012-09-25

摘要/Abstract

摘要： microRNA(miRNA)是一类细胞内源表达的小分子非编码RNA, 主要通过降解靶基因的mRNA或者抑制靶基因的翻译, 在动植物的发育以及其他重要的生理过程中起调控作用。miRNA的功能跟它的表达位置与时间密切相关, 但是目前尚缺乏一个能够在活体与个体水平稳定、持续地实时观察miRNA动态表达的方法。文章以斑马鱼为模式, 建立了一个双荧光报告系统(我们称之为miRNA Tracer), 用于在斑马鱼整体胚胎中追踪特定miRNA的表达谱及动态变化过程。该系统以Tol2转座子为基础, 采用来自斑马鱼hsp70基因的热激启动子分别驱动eGFP和mRFP1荧光报告基因, 同时在其中一个报告基因的3′-UTR区连接待测miRNA的互补序列, 构成Tracer质粒。该互补序列与斑马鱼胚胎中相应的内源miRNA结合后能够使对应报告基因的荧光信号强度减弱, 通过比较两个报告基因在表达谱上的差异辨别miRNA的表达区域, 检测斑马鱼胚胎中miRNA起作用的位置和时间。文章选择在肌肉系统特异表达的miR-206以及在神经系统特异表达的miR-219, 分别在显微注射瞬时表达和转基因稳定整合等两个层次上验证了上述Tracer系统。结果表明, 所用的方法能够如实地在单细胞水平和整体水平检测到目标miRNA的时空表达动态变化。miRNA Tracer系统为在斑马鱼发育过程中对miRNA进行活体、实时的时空定位提供了一个独特而有效的方法, 也为对miRNA进行功能与作用机制等更深入的研究奠定了基础。

补充资料

s219mRFP1-dF转基因胚胎的3-D图像 [视频]

关键词: 斑马鱼, miRNA, 双荧光报告载体/Tracer, 热激启动子

Abstract: 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.

Key words: zebrafish, microRNA, dual-fluorescence reporter/miRNA Tracer, heat shock promoter

杨红波，梁巍，刘新星，朱作言，林硕，张博. 应用双荧光报告系统检测斑马鱼microRNA的动态表达[J]. 遗传, 2012, 34(9): 1181-1192.

YANG Hong-Bo, LIANG Wei, LIU Xin-Xing, ZHU Zuo-Yan, LIN Shuo, ZHANG Bo. Detection of dynamic expression of microRNAs in vivo using a dual-fluorescence reporter system/miRNA Tracer in zebrafish[J]. HEREDITAS, 2012, 34(9): 1181-1192.

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应用双荧光报告系统检测斑马鱼microRNA的动态表达

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

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