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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.


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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