Inducible and Reversible Clock Gene Expression in Brain Using the tTA System for the Study of Circadian Behavior

Abstract

The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these ''clock genes'' are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus-and brain-directed expression of a tetO::Clock D19 dominantnegative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clock wt wild-type transgene shortened the period. Low doses (10 lg/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissuespecific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse.

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

“…Hong et al (50) reported that high doses of doxycycline can lead to prolonged suppression of Cre expression even after withdrawal of doxycycline, whereas lower doses of doxycycline allow improved titration of Cre expression. We therefore tested doxycycline at a 200-fold lower dose (10 g/ml) in drinking water starting from conception.…”

Adrenal peripheral clock controls the autonomous circadian rhythm of glucocorticoid by causing rhythmic steroid production

“…As with fibroblasts (19), a 2-h serum shock was able to synchronize the molecular clock in the Y1 cells. The serum shock caused rhythmic steroid production and cyclic StAR mRNA accumulation with Ϸ24-h periodicity that persisted for up to 60 h. These features clearly depended on an intact cellular clock, because overexpression of CLOCK⌬19, a defective mutant of CLOCK (20), dampened the cyclic profiles of both steroid production and StAR mRNA expression ( Fig. 2 A and B).…”

Peripheral Circadian Clocks in the Vasculature

“…Assessing cardiovascular parameters throughout the circadian day in transgenic mouse models with disrupted circadian clocks has provided initial information in this regard. 48,50 However, forthcoming studies of mouse models with tissue restricted dysfunctional molecular clocks 51,52 will allow a greater understanding of the relative contribution of both systemic cues and these peripheral oscillators to physiology and disease.…”