Detecting the Rhythms of Life with Synergy Readers
Dr. John Hogenesch
Most organisms have circadian clocks, which regulate 24- hour rhythms in a wide variety of activities and behaviors. In humans, the circadian clock regulates sleep onset and influences how much sleep you need. Not surprisingly, changes to these clock factors influence sleep, but also to a wide variety of disorders such as metabolic syndrome, cardiovascular disease, and even cancer. Dr. John Hogenesch, Ohio Eminent Scholar; Professor of Pediatrics in the Divisions of Human Genetics and Immunobiology and the Perinatal Institute; and Deputy Director, Center for Chronobiology at the Cincinnati Children's Hospital Medical Center, in Cincinnati, Ohio, is one of the world’s foremost experts in the molecular mechanisms of circadian rhythms in mammals. His research centers around understanding how biological clocks work and leveraging them to improve human health.
The Hogenesch laboratory uses several human and mouse cellular models of circadian clock function in which a promoter for a clock factor, such as BMAL1 or PER2, is fused to a luciferase reporter gene. Using these reporter cells, luminescence can be measured every 30 minutes for days or even weeks; tracking their own autonomous circadian rhythms. These rhythms can be measured by themselves or after treating with small molecules, siRNAs, cDNAs, or other genetic perturbations. For over a decade, the Hogenesch laboratory relied on BioTek’s Synergy™ 2 Multi-Mode Reader to conduct the luminescent measurements. In fact, several years ago, when laying the foundation to democratize luminescence-based medium throughput screening in chronobiology, Dr. Hogenesch and his colleague, Dr. Andrew Liu at the University of Florida developed reporter cell lines using the Synergy 2. This information was included in the 2014 PLOS Genetics paper, titled, “Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models” (doi: 10.1371/journal.pgen.1004244).
Recently, when Dr. Hogenesch decided to purchase an additional reader to keep up with the luminescence assay throughput demands, he once again turned to BioTek; this time for the Synergy Neo2 Hybrid Multi-Mode Reader. “Synergy Neo2 has the sensitivity and short reading time that we require for our samples,” notes Dr. Hogenesch. “Also, it’s critical to maintain constant temperature during luminescence measurements, which both Synergy readers do very well. Measuring in 96-well format is valuable, as it enables medium throughput genetic and small molecule screening” he adds. In addition to the performance, he notes that the constant reliability of the Synergy readers is particularly appreciated as it eliminates the risk of unplanned service costs and downtime. “I’d rather not be on a first name basis with a service tech, if you know what I mean,” said Dr. Hogenesch.
BioTek’s Synergy readers enjoy a reputation for robustness and dependability across the specialized chronobiology community, and Dr. Hogenesch cites several colleagues that use the readers in institutions such as the University of Pennsylvania’s Perelman School of Medicine, St. Jude Children’s Research Hospital, University of Florida, and the Morehouse School of Medicine.
Figure 1. Mammalian circadian luciferase reporter line. U2OS PER2:dLuc#41 (Hogenesch lab) stably express the Luciferase gene under the control of a minimal PER2 reporter containing only the E-box consensus sequence. Cells were synchronized with dexamethasone prior to the run in a luminescence recording medium. Wells were read at ~1 hr resolution for 6 days from the bottom optics of the Synergy Neo2 in a 96 well plate. Mean luminescence (RLU) +/- the standard deviation plotted (n=12).
Figure 2. Phenotype example from a 96-well plate screen using Synergy Neo2. Short period circadian phenotype (-3.2 hr, n=3/treatment).
To learn more about the Cincinnati Children's Hospital Medical Center web site.
Thanks to Dr. John Hogenesch for sharing his BioTek experience.