| 研究課題/領域番号 |
20K18366
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| 研究機関 | 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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研究代表者 |
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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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | Normal-tension glaucoma / splicing / causative gene |
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| 研究実績の概要 |
We identified a novel causative mutation of NTG in gene X by performing WES. Moreover, this novel mutation exhibits the gain of aberrant mRNA splicing that leads to retention of intros or skipping exon and eliminates full-length protein using several different cell culture models, including patient-derived iPSCs. The aberrantly spliced variants affected protein production and altered subcellular localization. Furthermore, we developed and characterized the knock-in and knockout mouse models. Both knock-in and knockout mice had decreased RGCs and retinal thickness. Last, we differentiated iPSCs into RGCs by inhibiting SMAD and Wnt pathways. The mutational effects of the novel gene were analyzed in purified iPSC-RGCs.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
1. We demonstrated this novel mutation exhibits the gain of aberrant mRNA splicing that leads to retention of intros or skipping exon and eliminates full-length protein using several different cell culture models, including patient-derived iPSCs.
2. The aberrantly spliced variants affected protein production and altered sub-cellular localization in three cell lines.
3. To gain further insight into the function of mutation, we analyzed GC complex thickness, disc & cup volume and number of GC in knock-in and knockout mice. B-circular and B-horizontal of OCT scan revealed that the thickness of RGC fiber layer around optic nerve head (ONH) significantly reduced and disc & cup volume increased dramatically in both knock-in and knockout mice compared with controls in 4-month and 6-month of age.
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| 今後の研究の推進方策 |
1. Eases the effects of the novel mutation on RGCs. We have documented decreased ganglion cell complex thickness and increased optic cup volume in knock-in and knockout mice. the effects of this mutation on RGCs will be confirm by robust retinal ganglion cell counts and RGC axon counts within the optic nerve.
robust retinal ganglion cell counts using flat mounts and PPD staining of retrobulbar optic nerve.
2. To strengthen the characterization of the knock-in and knockout mice model, the functional measure of RGC activity, such as pSTR, will be performed.
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| 次年度使用額が生じた理由 |
iPSC-RGCs differentiation proceeded through two phases: PRC induction and differentiation; RGC initiation and differentiation. Because RPCs are multipotent cells, they have the potential to differentiate into neuronal cell types and one glial cell type called the Muller glial cells. Therefore, we need to purify iPSC-RGCs by Thy1 cell surface marker using CD90.2 microbeads.
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