2015 Fiscal Year Research-status Report
フォワード・ジェネティックスを用いた先天的恐怖の分子メカニズム解明
Project/Area Number |
15K14874
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Research Institution | University of Tsukuba |
Principal Investigator |
Liu Qinghua 筑波大学, 国際統合睡眠医科学研究機構, 教授 (90723792)
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Co-Investigator(Kenkyū-buntansha) |
曹 麗琴 筑波大学, 国際統合睡眠医科学研究機構, 助教 (60399475)
クレウエ・ネベニウス ダニエラ 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (60737667)
佐藤 牧人 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (70743699)
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Project Period (FY) |
2015-04-01 – 2017-03-31
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Keywords | 恐怖情動 / 先天的恐怖 |
Outline of Annual Research Achievements |
Mice mainly rely on a very sensitive olfactory system to detect predators. Thus, we developed a novel predator odor-based “innate fear” assay that is suitable for high throughput mouse screening. In this 20 minute assay, TMT (2,3,5-trimethyl-3-thiazoline), a component of fox feces, induces robust (innate fear) freezing response in mice. Using “Freezeframe” software to quantify freezing as a readout of fear, wild-type mice freeze 0-20% without odor and 60-95% time in response to TMT. This robust assay has a tight relative standard deviation (RSD) of ~10%, making it feasible to screen for “fearless” mutants with <47% freezing rate [i.e. >3 SD below normal mean of 77%]. To date, we screened 1,596 F1 (c57 B6J/B6N) ENU mutant males and identified ten phenodeviants that showed significantly diminished freezing rate. We established two dominant mutant pedigrees: whereas one showed the “fearless” phenotype, the other mutant called “Popcorn” exhibited an unusual phenotype of jumping up to ~2,500 times/20 minutes when exposed to the predator odor. By single polynucleotide polymorphism (SNP) linkage analysis, we mapped the Popcorn mutation to a 70Mb critical interval on Chromosome 1 (p<10^-15). Exome sequencing of wild-type and mutant mice revealed a candidate causal mutation in a novel gene. We propose that Popcorn is a putative “fearful” mutant, which may be genetically predisposed to fear/anxiety disorders in a manner similar to the “onco(gene) mice” that develop cancer with age.
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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
We worked very diligently and have excellent help from outstanding collaborators: 1) We collaborate with Drs. Ko and Reiko Kobayakawa, whom are pioneers in the innate fear research, to establish the predator odor-induced innate fear assay. 2) We used the ENU mutant mice generated by Dr. Masashi Yanagisawa for his sleep screen. His extensive experience in forward genetics help expedite our screen. 3) We collaborated with Dr. Shigeharu Wakana, who has many years of experience in genetic mapping, to map the Popcorn mutation. 4) We performed exome sequencing in the Next Generation Sequencing (NGS) core at UT Southwestern Medical Center, USA to identify the Popcorn mutation.
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Strategy for Future Research Activity |
1) To re-create Popcorn mutant mice by CRISPR to verify the causality of Popcorn mutation. 2) To examine expression pattern of Popcorn gene in mouse brain by in situ hybridization. 3) To perform whole brain c-fos mapping to map where Popcorn may act in the brain. 4) To determine whether Popcorn is a “fearful” mutant by physiological and behaviors assays.
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