Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2024;2805:137-151. doi: 10.1007/978-1-0716-3854-5_9.
Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.
在发育中的后生动物中，转录本质上是随机的，涉及转录机制之间的瞬时和动态相互作用。传统技术（包括固定组织的蛋白质和 RNA 染色）的一个基本挑战是缺乏时间分辨率。量化转录的动力学变化可以阐明调控模块之间相互作用的潜在机制。在本方案中，我们描述了成功实施 MS2/MCP 和 PP7/PCP 系统组合在活体果蝇胚胎中的应用，以进一步了解发育过程中的转录动力学。我们的技术可以扩展到同时可视化多个基因或等位基因的转录活性，表征靶基因的等位基因特异性表达，并以单细胞分辨率定量分析 RNA 聚合酶 II 的活性。
