title: An improved method for the study of apoptosis-related genes using the tet-on system.
abstract: 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.
authors.affiliation: Department of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center, Rochester, NY 14642, USA. marc_halterman@urmc.rochester.edu
authors.forename: Marc W
authors.initials: MW
authors.lastname: Halterman
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D000860:Hypoxia / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic / Q000187:drug effects; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D053148:Caspase 3; D013752:Tetracycline; D004318:Doxycycline