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J Biomol Screen. 2011 Mar;16(3):332-7. doi: 10.1177/1087057110397355. Epub 2011 Feb 18.

An improved method for the study of apoptosis-related genes using the tet-on system.

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  • 1Department of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center, Rochester, NY 14642, USA. marc_halterman@urmc.rochester.edu


Inducible gene expression systems are particularly useful for the functional characterization of genes with putative toxic properties. In the course of studying the role of hypoxia-regulated gene expression on cell survival using the tetracycline-inducible (tet-on) system, the author noted that exposure to the inducing ligand doxycycline (dox) inhibited caspase-3 cleavage in control samples. To limit this confounding off-target effect, he devised an in vitro pulse dose, delayed-injury protocol testing both dox and a novel tetracycline analog 9-t-butyl doxycycline (9-TB). Although 9-TB induced higher transgene levels compared to matched concentrations of dox, continuous exposure to both drugs inhibited caspase-3 cleavage in hypoxic samples. Conversely, a 6-h pulse dose of 9-TB followed by a 40-h washout period prior to hypoxic challenge activated robust transgene expression and lessened the inhibitory effects on caspase-3 processing. It is anticipated that these protocol modifications will improve the performance of tet-regulated genetic screens, particularly in situations where cell death is used as a primary end point.

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