| Project/Area Number |
23K15923
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 56060:Ophthalmology-related
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| Research Institution | 独立行政法人国立病院機構(東京医療センター臨床研究センター) |
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Principal Investigator |
潘 洋 独立行政法人国立病院機構(東京医療センター臨床研究センター), 分子細胞生物学研究部, 研究員 (20866389)
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| Project Period (FY) |
2023-04-01 – 2026-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2025: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2024: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2023: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | OMD / オカルト黄斑ジストロフィ / 遺伝子 / 発症分子メカニズム / iPS細胞 |
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| Outline of Research at the Start |
オカルト黄斑ジストロフィ（OMD）は網膜中心部(黄斑部)の錐体細胞の機能が低下し、次第に視力が低下する疾患である。患者の多くは遺伝性であり、まだ根本的な治療法が存在しない。我々は顕性遺伝OMDの原因遺伝子がRP1L1であることを同定し、加えてRP1L1変異の潜性遺伝による錐体ジストロフィや網膜変性を紹介してきた。RP1L1は網膜視細胞における重要な生理的役割を担うことを示唆されており、本研究ではRP1L1正常体と変異体のタンパク質機能を比較し、家系内の患者および健常者のiPS細胞から視細胞を分化誘導して、発症分子メカニズムの解明をめざす。得られた知見に基づき、OMDの新規分子標的の探索を行う。
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| Outline of Annual Research Achievements |
1. Establishment of iPSCs:
1) Lymphocytes were collected from both patients and healthy individuals within the same family lineage. 2) After culturing, iPS cells were established using Sendai virus vectors containing Yamanka induction factors.
2. Differentiation into photoreceptor-like cells:
1. iPSCs were cultured in a feederless system and transfected with CRX and NEUROD1 genes using a piggyBac vector. 2) Stable iPSC clones were selected and confirmed for gene expression using PCR and RT-qPCR. 3) These cells were then induced to differentiate into photoreceptor-like cells by adding doxycycline to activate gene expression. 4) The markers of photoreceptor cells were analyzed, demonstrating successful differentiation and potential applications in vision-related research and therapies.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
1. Our research was guided by precise objectives, centered on establishing iPS cells and their subsequent differentiation into photoreceptor-like cells. This clarity facilitated the establishment of clear milestones and provided a definitive direction for our work.
2. Before each experimental step, we engaged in meticulous planning, ensuring that protocols were followed rigorously and potential issues were anticipated and addressed proactively.
3. Throughout the research process, we implemented stringent quality control measures at various stages. This included the careful selection of stable iPSC clones and thorough confirmation of gene expression, which played a crucial role in upholding the integrity and reliability of our research outcomes.
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| Strategy for Future Research Activity |
1. Functional Characterization in Transfected Cells (COS-7 and HEK293T):
1)Transfect COS-7 and HEK293T cells. 2) Perform WB to assess expression levels of the mutated gene. 3) Conduct immunofluorescence assays to examine intracellular localization of the mutated protein.
2. Mutation Effects on Protein Interaction:
1) Investigate the impact of the mutation on protein-protein interactions. 2) Utilize WB and immunoprecipitation techniques to evaluate changes in protein interactions caused by the mutation.
3. Conduct RNA sequencing analysis on iPRCs.
1) Analyze gene expression patterns to understand the effects of the mutation on cellular processes. 2) Validate changes observed in RNA sequencing through RT-qPCR and WB analysis.
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