2017 Fiscal Year Annual Research Report
フォワード・ジェネティクスで同定した新規睡眠制御遺伝子による分子機構の解明
| Project/Area Number |
17J07957
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| Research Institution | University of Tsukuba |
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Principal Investigator |
KIM StaciJakyong 筑波大学, グローバル教育院, 特別研究員(DC2)
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| Project Period (FY) |
2017-04-26 – 2019-03-31
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| Keywords | Forward genetics / Sleep |
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| Outline of Annual Research Achievements |
Sleepy2 was identified throughout the forward genetics screening of sleep and wake behavior. Highly conserved Sleepy2 gene family may play shared role in the regulation of sleep and wake. To understand the link among the Sleepy2 family genes as well as their specific role in sleep and wake modulation, we established mutant mouse lines in the C57BL/6 background using the CRISPR/Cas9 system. Sleepy2II and Sleepy2IV, also showed decrease in the total wake time with more pronounced change during the dark phase.
Also, three brain-specific conditional knockout mouse lines were engineered by Cre-loxP recombination. Each of the mutant mice pedigree were examined by EEG/EMG-based polysomnography analysis to assess any changes in their sleep and wake behavior.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
To examine constitutive role of Sleepy2 gene family in sleep and wake regulation, we created three additional mutant mouse lines by CRISPR/Cas9 system in Sleepy2 gene family: Sleepy2II, Sleepy2III, and Sleepy2III. The EEG/EMG-based polysomnography analysis showed changes in the sleep physiology in all three family member mutants. We chose Sleepy2II and Sleepy2IV to cross with Sleepy2 creating double-hetero mutant mice. The EEG/EMG-based polysomnography analysis revealed enhanced perturbation in the sleep and wake behavior in the double-hetero mutants when compared to the ancestral single mutants.
In order to understand the molecular mechanism of sleep need that drives the sleep behavior, changes in gene transcription can be examined by the next-generation sequencing. We completed optimization of the experimental condition and design with the preliminary run.
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| Strategy for Future Research Activity |
The molecular basis of sleep and wake behavior, specifically focusing on how sleep homeostasis is maintained and controlled, is still not well understood. We will assess transcriptome changes in various vigilance states. The sleep need increases during waking, and further enhanced when the animal is deprived of normal sleep as well as by Sleepy2 mutation. Brain samples of wild type and mutant mice will be taken during normal sleep, after sleep-deprivation, and during spontaneous wake period. For those regions of the brain showing differential expression, quantitative PCR will be used to determine whether the changes in the gene expression occurs in the entire brain or locally.
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