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.
The reaction of anilines bearing a benzylic activating group in the ortho position with aromatic aldehydes or a,b-unsaturated aldehydes results in an efficient synthetic route to substituted indoles.
Changes in gene expression play a central role in determining the physiological activities of cells. To understand the dynamics of gene expression several techniques have been developed recently but their applicability have been limited by factors like choice of reporter and target selection. We have developed an aptamer‐based imaging system to detect changes in promoter activity in living cells and in real time. In this technology we have cloned and expressed multiple copies of aptamers in tandem (viz. tobramycin or neomycin‐B or PDC) under inducible or constitutive promoters in Saccharomyces cerevisiae. The yeast cells were incubated with the respective ligands, which were conjugated to a Cy3 or a Cy5 dye. The promoter activities were then monitored by FRET (Förster resonance energy transfer), which occurred due to binding of the multiaptamers to the fluorophore labeled ligands. This technology allows the visualization of real time promoter activity from an inducible promoter like GAL1 by FLIM‐FRET, which was detected by the decrease of donor lifetime. We have been able to measure promoter activity from individual cells and found intercellular variation of gene expression upon induction. The IMAGEtag technology has the potential for application in measuring gene expression during various cellular events and for understanding it's distribution and temporal change in individual cells of the same type within multicellular or homogeneous populations.Funded by National Institute of Biomedical Imaging and Bioengineering, U.S. Department of Energy, Office of Biological and Environmental Research
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