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Christina Winborn, Timothy E. McKnight, John C. Lang, Edward Chaum; Nanoscale Probes and Platforms for Analysis of Gene Expression in Living RPE Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):272.
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Molecular beacons (MB), self-quenching hairpin-shaped oligonucleotides that fluoresce upon hybridization to target mRNA molecules, have recently been used to detect gene expression in living cells. The goal of these studies was to utilize MBs to quantify gene expression in retinal pigment epithelial cells (RPE). We quantified expression of both an IGF1 transgene and oxidative stress-induced (OS) FOS expression in the RPE in vitro. We also examined strategies for tethering MBs to carbon nanofiber (CNF) arrays, nanostructured cell-penetrating platforms with the potential to signal gene expression in living cell monolayers.
We designed and synthesized MBs for FOS, IGF1, GAPDH, and a non-binding negative control. MBs were delivered into RPE cells via linkage to the cell penetrating peptide TAT1. Gene expression (as seen by MB fluorescence) was quantified using confocal image analysis and Nikon EZC1 software. We quantified MB fluorescence in IGF1-transfected cells relative to naïve RPE cells with low intrinsic IGF1 expression. We also quantified OS-induced FOS gene expression in RPE cells exposed to OS. Finally, we tethered IGF1 or FOS MBs to a CNF array and quantified focal fiber fluorescence in the presence of complimentary oligonucleotides.
We observed high IGF1-specific MB fluorescence in RPE cells stably transfected with IGF1, 11- fold greater than fluorescence seen in naïve RPE cells. Similarly, FOS MB fluorescence increased 9-fold (compared to controls) in cells exposed to OS. These quantitative changes in gene expression were validated by qRT-PCR performed in replicate RPE cell cultures. Finally, CNF arrays with tethered IGF1 and FOS MBs show focal fiber fluorescence in the presence of complimentary oligonucleotides, demonstrating that they can be used as nanoscale platforms to signal gene expression in living cell monolayers.
Molecular beacons are novel nanoscale probes that can be used to detect and quantify gene expression in living RPE cells under a variety of experimental paradigms.
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