title: Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ilchung
authors.initials: I
authors.lastname: Shin
authors.affiliation: 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.
authors.forename: Judhajeet
authors.initials: J
authors.lastname: Ray
authors.affiliation: Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
authors.forename: Vinayak
authors.initials: V
authors.lastname: Gupta
authors.affiliation: 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.
authors.forename: Muslum
authors.initials: M
authors.lastname: Ilgu
authors.affiliation: Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Beasley
authors.affiliation: Ames Laboratory, US Department of Energy, Ames, IA 50011, USA.
authors.forename: Lee
authors.initials: L
authors.lastname: Bendickson
authors.affiliation: Molecular Express, Inc., Ames, IA 50014, USA.
authors.forename: Samir
authors.initials: S
authors.lastname: Mehanovic
authors.affiliation: Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
authors.forename: George A
authors.initials: GA
authors.lastname: Kraus
authors.affiliation: 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 marit@iastate.edu.
authors.forename: Marit
authors.initials: M
authors.lastname: Nilsen-Hamilton
mesh_terms: D052157:Aptamers, Nucleotide; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D052157:Aptamers, Nucleotide; D012319:RNA Polymerase II
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