Inducible and Reversible Clock Gene Expression in Brain Using the tTA System for the Study of Circadian Behavior
Hee-Kyung Hong1,2,3, Jason L. Chong3, Weimin Song1,3, Eun Joo Song1,3, Amira A. Jyawook2,3, Andrew C. Schook1,3, Caroline H. Ko3,4, Joseph S. Takahashi1,2,3*
1 Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois, United States of America, 2 Center for Functional Genomics, Northwestern University, Evanston, Illinois, United States of America, 3 Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, United States of America, 4 Department of Psychology, University of Toronto, Toronto, Ontario, Canada
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::ClockΔ19 dominant-negative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clockwt wild-type transgene shortened the period. Low doses (10 μg/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 tissue-specific, 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.
Funding. This work was supported by Silvio O. Conte Center National Institutes of Health grant P50 MH074924 to JST. JST is an investigator and HKH was an associate of the Howard Hughes Medical Institute.
Competing interests. The authors have declared that no competing interests exist.
Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America
Abbreviations: AVP, arginine vasopressin; BAC, bacterial artificial chromosome; CT, circadian time; DD, constant darkness; Dox, doxycycline; ENU, N-ethyl-N-nitrosourea; HA, hemagglutin; PRC, phase-response curve; SCN, suprachiasmatic nucleus; TG, transgene; tetO, tet operator; tTA, tetracycline-controlled transactivator; VIP, vasoactive intestinal polypeptide; WT, wild-type
* To whom correspondence should be addressed. E-mail: [email protected]
Although significant progress has been made in unraveling the molecular mechanism of circadian clocks in mammals, previous work has focused on germline mutations and in vitro methods for analysis. To address the function of clock genes, it is necessary to develop tools to manipulate circadian genes in a conditional and tissue-specific manner in vivo. We report such an approach using the tetracycline transactivator system. Despite the development of the “tet” system in transgenic mice over 10 y ago by Bujard and colleagues, there are still relatively few examples of the successful use of the tet system in the central nervous system. Transgenic expression of the Clock gene in the suprachiasmatic nucleus and brain of mice regulated the period length of circadian locomotor rhythms. These effects could be inhibited by low doses of doxycycline in the drinking water. Importantly, low, but not high, doses of doxycycline were completely reversible and led to a rapid reactivation of the Clock transgenes. In summary, the tetracycline-controlled transactivator system can manipulate circadian clock gene expression in a tissue-specific, 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.
PLoS Genet 3(2): e33. This is an open-access article distributed under the terms of the Creative Commons Attribution License.