| Project/Area Number |
16K11810
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Research Field |
Orthodontics/Pediatric dentistry
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| Research Institution | Kagoshima University |
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Principal Investigator |
Kubota Naoko 鹿児島大学, 医歯学域歯学系, 助教 (40569810)
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| Co-Investigator(Kenkyū-buntansha) |
佐藤 正宏 鹿児島大学, 総合科学域総合研究学系, 教授 (30287099)
稲田 絵美 鹿児島大学, 医歯学域附属病院, 助教 (30448568)
野口 洋文 琉球大学, 医学(系)研究科(研究院), 教授 (50378733)
齊藤 一誠 新潟大学, 医歯学系, 准教授 (90404540)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2017: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | 生体内遺伝子導入 / PiggyBacシステム / CRISPR/Cas9 / piggybacシステム |
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| Outline of Final Research Achievements |
The EGFP cDNA-expressing piggyBac transposon vector was directly injected into the tissue around the root forming apex of juvenile mice using our newly-developed technique. In vivo electroporation was then immediately performed at the injected sites. We observed sustained EGFP expression six weeks after gene delivery. The introduced transgene was detectable via PCR using genomic DNA isolated from the injected tissues. This piggyBac-based gene delivery system was also found to be useful for application in primary cultured human deciduous teeth-derived dental pulp cells and allowed long-term expression of transgenes and efficient acquisition of stable transfectants. Application of a genome editing system (CRISPR/Cas9) designed to destroy endogenous target genes was also successful in mice and cultured cells. These results could facilitate the application of genetic engineering in the dental field.
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| Academic Significance and Societal Importance of the Research Achievements |
「遺伝子工学的手法」を用い、生体内歯周組織への効率的な遺伝子導入法の検討は、歯科領域では未だ十分に検討されていない。更に、単に一過的な遺伝子発現で満足するのではなく、外来性遺伝子の宿主染色体への効率的な組み込みを可能とするpiggyBacトランスポゾン系を応用し、外来性遺伝子を導入組織に定着させ、遺伝子の持続的発現を試みることは、独創性と新規性がある。その結果、「生体内での外来性遺伝子の宿主染色体への挿入による持続的遺伝子発現の達成」、「最近開発されたゲノム編集による標的遺伝子破壊の可能性」を示したことは、「歯科領域における生体内遺伝子工学」という新たな流れを作った点で学術的意義がある。
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