2023 Fiscal Year Research-status Report
RP1L1遺伝子とそのオカルト黄斑ジストロフィ関連変異体の分子機能解析
| Project/Area Number |
23K15923
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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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| Keywords | OMD |
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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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| Causes of Carryover |
The remaining funds could be due to various factors, such as completing project phases under budget, efficient resource management, or unexpected savings from procurement activities.
Usage plan with carryover funds: the carryover funds will be strategically utilized in alignment with the objectives and activities outlined in the grant proposal for the following fiscal year. The plan includes the continuation of research activities, enhancement of project scope, dissemination, and outreach.
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[Journal Article] 正常眼圧緑内障の新しい原因遺伝子METTL23を同定2023
Author(s)
潘 洋, 須賀 晶子, 木村 至, 木村 肇二郎, 峯岸 ゆり子, 中山 真央, 吉武 和敏, 家島 大介, 峰松 尚子, 山本 めぐみ, 間渕 文彦, 高本 光子, 志賀 由己浩, 新家 眞病, 柏木 賢治, 相原 一, 中澤 徹, 岩田 岳
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Journal Title
日眼会誌
Volume: 127 (4)
Pages: 507
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