In vivo modulation and quantification of microRNAs during axolotl tail regeneration.
In vivo modulation and quantification of microRNAs during axolotl tail regeneration.
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DOI：
10.1007/978-1-4939-2495-0_13
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Echeverri K
中科院分区：
文献类型：
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作者：
Erickson JR;Echeverri K
再生患病、受伤或缺失的复杂组织的能力在低等脊椎动物和无脊椎动物中广泛存在;然而，我们对调节这种惊人能力的分子机制的认识仍处于起步阶段。最近的许多论文已经显示了microRNA在许多物种中调节再生的重要作用。在体内不同细胞类型的单个细胞水平上检测和定量miRNA表达波动的能力在再生过程中是非常有用的。在本章中，我们描述了如何使用双荧光绿色荧光蛋白（GFP）-报告基因/单体红色荧光蛋白（mRFP）-传感器（DFRS）质粒来定量特定miRNA在miRNA模拟物注射后以及再生过程中随时间的动态变化。在该双顺反子载体中，mRFP允许验证miRNA表达，而GFP用作内部对照以使miRNA表达标准化，从而获得定量结果。此外，我们还展示了这项技术如何揭示尾部再生过程中miR-23 a的动态表达和功能。
The ability to regenerate diseased, injured, or missing complex tissue is widespread throughout lower vertebrates and invertebrates; however, our knowledge of the molecular mechanisms that regulate this amazing ability is still in its infancy. Many recent papers have shown important roles for microRNAs in regulating regeneration in a number of species. The ability to detect and quantify miRNA expression fluctuations at a single cell level in vivo in different cell types during processes like regeneration is very informative. In this chapter, we describe how to use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time following miRNA mimic injection as well as during regeneration. In this bicistronic vector, the mRFP allows for verification of miRNA expression, while the GFP functions as an internal control to normalize miRNA expression and thus obtain quantitative results. In addition, we demonstrate how this technique revealed dynamic miR-23a expression and function during tail regeneration.