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Measuring the Kinetics of mRNA Transcription in Single Living Cells
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测量活细胞内部个体基因的转录动态
Ineke Brouwer1, Marit A C de Kort1, Tineke L Lenstra2
1Division of Gene Regulation, the Netherlands Cancer Institute, Oncode Institute, Amsterdam, the Netherlands.
Methods in molecular biology (Clifton, N.J.)
|October 12, 2023
概括
本研究详细介绍了使用MS2和PP7RNA标记技术在单细胞中可视化基因转录突发的方法. 这些方法可以量化酵母和哺乳动物细胞中的转录动态和爆裂动态.
科学领域:
- 分子生物学分子生物学
- 细胞生物学 细胞生物学
- 遗传学 是一个遗传学.
背景情况:
- 基因转录是一个动态的过程,经常发生在随机爆发.
- 了解这些爆发的调节需要实时观察单细胞的转录.
研究的目的:
- 描述可视化和量化单个活细胞转录动态的方法.
- 为了研究内源基因的转录突破动力学.
主要方法:
- 使用MS2和PP7RNA标记技术,以光标记单个基因的新生转录.
- 使用活细胞单分子显微镜进行实时转录观测.
- 用MS2/PP7系统对发芽酵母 (Saccharomyces cerevisiae) 和哺乳动物细胞 (老鼠胚胎干细胞) 进行基因工程.
主要成果:
- 在酵母和哺乳动物细胞中成功实施MS2和PP7系统.
- 建立实时显微镜实验的协议.
- 开发用于提取转录突破参数的程序.
结论:
- 描述的方法为研究单细胞水平的转录动态提供了一个强大的框架.
- 这种方法促进了对基因调节机制的研究,这些机制是转录突破的基础.
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