Imaging promoter activity to monitor gene expression using Intracellular Multiaptamer Genetic tag (IMAGEtag)

Abstract

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