The dynamics of gene expression are fundamental to the coordination of cellular responses. Measurement of temporal gene expression patterns is currently limited to destructive lowthroughput techniques such as northern blotting, reverse transcription polymerase chain reaction (RT-PCR), and DNA microarrays. We report a scalable experimental platform that combines microfluidic addressability with quantitative live cell imaging of fluorescent protein transcriptional reporters to achieve real-time characterization of gene expression programs in living cells. Integrated microvalve arrays control row-seeding and column-stimulation of 256 nanoliter-scale bioreactors to create a high density matrix of stimulus-response experiments. We demonstrate the approach in the context of hepatic inflammation by acquiring ~5000 single-time-point measurements in each automated and unattended experiment. Experiments can be assembled in hours and perform the equivalent of months of conventional experiments. By enabling efficient investigation of dynamic gene expression programs, this technology has the potential to make significant impacts in basic science, drug development, and clinical medicine.
The purpose of this study was to determine the kinetics of HSP70 expression in response to mild thermal stress. The rationale is to produce a basis for design of optimal heating methods to induce HSP70 expression for preconditioning in cardiac surgery. Bovine aortic endothelial cells were heated at 42 degrees C for 0.5 to 5 hours followed by 37 degrees C recovery for 1 to 48 hours. Quantitative analysis of western blot results showed HSP70 expression kinetics is a coupled function of heating temperature and time and of post-heating duration. Bimodal HSP70 expression kinetics were identified which may be an important cause of the "second window of protection" observed by other researchers.
Thermal preconditioning protocols for cardiac cells were identified which produce elevated HSP70 levels while maintaining high cell viability. Bovine aortic endothelial cells were heated with a water bath at temperatures ranging from 44 to 50 degrees C for periods of 1-30 min. Thermal stimulation protocols were determined which induce HSP70 expression levels ranging from 2.3 to 3.6 times the control while maintaining cell viabilities greater than 90%. An Arrhenius injury model fit to the cell damage data yielded values of A = 1.4 X 10(66) s(-1) and Ea = 4.1 X 10(5) J/mol. Knowledge of the injury parameters and HSP70 kinetics will enhance dosimetry guideline development for thermal stimulation of heat shock proteins expression in cardiac tissue.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.