Shin Ilchung, Ray Judhajeet, Gupta Vinayak, Ilgu Muslum, Beasley Jonathan, Bendickson Lee, Mehanovic Samir, Kraus George A, Nilsen-Hamilton Marit
Ames Laboratory, US Department of Energy, Ames, IA 50011, USA Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, 1210 Molecular Biology Building, Iowa State University, Ames, IA 50011, USA.
Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Nucleic Acids Res. 2014 Jun;42(11):e90. doi: 10.1093/nar/gku297. Epub 2014 Apr 20.
We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.
我们描述了一种用于基因表达活细胞成像的核糖核酸（RNA）报告系统，以检测单个细胞中单个启动子上聚合酶II活性的变化。这些报告基因使用构成IMAGEtags（细胞内多适配体基因标签）的RNA适配体串，其可从任选的启动子表达。为了成像，将细胞与分别与FRET对Cy3和Cy5之一偶联的配体一起孵育。IMAGEtags在酵母中由GAL1、ADH1或ACT1启动子表达。在活细胞中对来自所有三个启动子的转录进行成像，并在添加半乳糖后随时间观察到GAL1启动子的转录增加。IMAGEtags的表达不影响细胞增殖或内源基因表达。该方法的优点是细胞中不产生可能有毒或随着积累以其他方式影响细胞反应的外源蛋白，IMAGEtags寿命短且产生信号不需要氧气。IMAGEtag RNA报告系统提供了一种实时追踪活细胞中转录活性变化的方法。
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