A synthetic low-frequency mammalian oscillator.
Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives.
Tigges
Marcel
M
Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
Dénervaud
Nicolas
N
Greber
David
D
Stelling
Joerg
J
Fussenegger
Martin
M
D000818:Animals; D016466:CHO Cells; D002940:Circadian Rhythm / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000032:analysis; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D019076:Transgenes
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators