Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.
Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.
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DOI：
10.1093/nar/gku297
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发表时间：
2014-06
影响因子：
14.9
通讯作者：
Nilsen-Hamilton M
中科院分区：
文献类型：
--
作者：
Shin I;Ray J;Gupta V;Ilgu M;Beasley J;Bendickson L;Mehanovic S;Kraus GA;Nilsen-Hamilton M
我们描述了一个核糖核酸（RNA）报告系统，用于基因表达的活细胞成像，以检测单个细胞中单个启动子上聚合酶II活性的变化。报告者使用一系列RNA适体，这些适体构成细胞内多适体遗传标签（IMAGEtags），可以通过选择的启动子表达。为了成像，细胞与它们的配体一起孵育，这些配体分别与FRET对之一Cy3和Cy5偶联。IMAGEtags在酵母中分别从GAL1、ADH1或ACT1启动子中表达。在活细胞中对所有三个启动子的转录进行成像，并在添加半乳糖后观察到GAL1启动子的转录增加。IMAGEtags的表达不影响细胞增殖和内源基因的表达。这种方法的优点是，在细胞中不会产生可能有毒或以其他方式影响细胞反应的外来蛋白质，IMAGEtags的寿命很短，并且不需要氧气来产生它们的信号。IMAGEtag RNA报告系统提供了一种在活细胞中实时跟踪转录活性变化的方法。
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.
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DOI：
10.1126/science.1202142
发表时间：
2011-04-22
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Larson DR;Zenklusen D;Wu B;Chao JA;Singer RH
通讯作者：
Singer RH
DOI：
10.1126/science.1207339
发表时间：
2011-07-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Paige JS;Wu KY;Jaffrey SR
通讯作者：
Jaffrey SR
影响因子：
3.7
作者：
Koelsch, Kristi A.;Wang, YuJing;Wren, Jonathan D.
通讯作者：
Wren, Jonathan D.
影响因子：
4.5
作者：
Hogg, J. Robert;Collins, Kathleen
通讯作者：
Collins, Kathleen
DOI：
10.1039/978184973251200001
发表时间：
2011-01-01
期刊：
STRUCTURAL AND CATALYTIC ROLES OF METAL IONS IN RNA
影响因子：
--
作者：
Auffinger, Pascal;Grover, Neena;Westhof, Eric
通讯作者：
Westhof, Eric