Department of Molecular, Cellular and Developmental Biology and BioFrontiers Institute, University of Colorado, Boulder, CO, USA.
Skelet Muscle. 2013 Aug 1;3(1):19. doi: 10.1186/2044-5040-3-19.
microRNA regulation plays an important role in the remodeling that occurs in response to pathologic and physiologic stimuli in skeletal muscle. In response to stress, microRNAs are dynamically regulated, resulting in a widespread "fine-tuning" of gene expression. An understanding of this dynamic regulation is critical to targeting future therapeutic strategies. Experiments elucidating this dynamic regulation have typically relied on in vitro reporter assays, ex vivo sample analysis, and transgenic mouse studies. Surprisingly, no experimental method to date allows rapid in vivo analysis of microRNA activity in mammals.
To improve microRNA studies we have developed a novel reporter assay for the measurement of skeletal muscle microRNA activity in vivo. To minimize muscle damage, hydrodynamic limb vein injection was used for the introduction of plasmid DNA encoding bioluminescent and fluorescent reporters, including click-beetle luciferase and the far-red fluorescent protein mKATE. We then applied this technique to the measurement of miR-206 activity in dystrophic mdx4cv animals.
We found that hydrodynamic limb vein injection is minimally damaging to myofibers, and as a result no induction of muscle-specific miR-206 (indicative of an injury response) was detected. Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups. Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.
Hydrodynamic limb vein injection of plasmid DNA followed by in vivo bioluminescent imaging is a novel assay for the detection of reporter activity in skeletal muscle in vivo. We believe that this method will allow for the rapid and precise detection of both transcriptional and post-transcriptional regulation of gene expression in response to skeletal muscle stress. Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.
微小 RNA 的调控在骨骼肌肉对病理和生理刺激的重塑过程中起着重要作用。在应激反应中，微小 RNA 被动态调控，导致基因表达的广泛“微调”。理解这种动态调控对于靶向未来的治疗策略至关重要。阐明这种动态调控的实验通常依赖于体外报告基因检测、离体样本分析和转基因小鼠研究。令人惊讶的是，迄今为止，还没有实验方法可以快速在哺乳动物体内分析微小 RNA 的活性。
为了改进微小 RNA 的研究，我们开发了一种新的报告基因检测方法，用于测量哺乳动物骨骼肌微小 RNA 的活性。为了最大限度地减少肌肉损伤，采用流体动力学肢体静脉注射法将编码生物发光和荧光报告基因的质粒 DNA 导入，包括荧光素酶和远红荧光蛋白 mKATE。然后，我们将该技术应用于测量肌营养不良症 mdx4cv 动物的 miR-206 活性。
我们发现流体动力学肢体静脉注射对肌纤维的损伤极小，因此没有检测到肌肉特异性 miR-206 的诱导（表明存在损伤反应）。与肌肉内注射或电穿孔不同，我们发现流体动力学肢体静脉注射导致报告基因在多个后肢肌肉群中呈分散表达。此外，我们利用来自 Pyrophorus plagiophthalamus 的荧光素酶作为报告基因，并用远红荧光蛋白 mKATE 进行归一化，作为原理证明，我们可以检测到 mdx4cv 动物的 miR-206 活性升高，与 C57Bl/6 对照相比。
质粒 DNA 的流体动力学肢体静脉注射，然后进行体内生物发光成像，是一种用于检测骨骼肌体内报告基因活性的新方法。我们相信，这种方法将允许快速、精确地检测骨骼肌应激反应中基因表达的转录和转录后调控。此外，由于肌纤维的有丝分裂后状态和质粒 DNA 的稳定表达，我们相信这种方法将通过允许对同一动物队列进行纵向研究，减少动物研究中的生物学变异性。
