In Vivo Modulation and Quantification of microRNAs During Axolotl Tail Regeneration
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.
Key wordsmicroRNAs Axolotl Sensor plasmids
We thank Davide De Pietri Tonelli and Antonio Giraldez for the kind gift of the DFRS plasmids.
The authors are grateful for support from the Stem Cell Training Grant (T32 HD060536 04) for this project as well as funding from the Department of Genetics, Cell Biology and Development at the University of Minnesota.
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