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KAKEN_16K11305seika.pdf
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Download
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:263.6 KB
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| Last updated |
:Oct 31, 2019 |
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Total downloads since Oct 31, 2019 : 575
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患者iPS細胞由来視細胞標識による網膜色素変性症疾患メカニズムの解析
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カンジャ iPS サイボウ ユライ シサイボウ ヒョウシキ ニ ヨル モウマク シキソ ヘンセイショウ シッカン メカニズム ノ カイセキ
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Kanja iPS saibō yurai shisaibō hyōshiki ni yoru mōmaku shikiso henseishō shikkan mekanizumu no kaiseki
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Investigation of disease mechanisms by fluorescent labeling of photoreceptor derived from retinitis pigmentosa patient-derived induced pluripotent stem cells
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本間, 耕平
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ホンマ, コウヘイ
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Honma, Kōhei
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慶應義塾大学・医学部 (信濃町)・特任助教
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Research team head
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科研費研究者番号 : 80462729
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2019
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科学研究費補助金研究成果報告書
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2018
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本研究では，これまでに既に作製された網膜色素変性症 (RP) 患者由来iPS細胞 (RP1，RP9，PRPH2，RHOの変異株) を使用した。得られたRPのiPS細胞のラインのゲノムを抽出し遺伝子変異を確認した。これらのラインから分化させた桿体視細胞を選択的に標識するために、ゲノム編集によるノックイン を行った。これによりGFPの蛍光で分化した視細胞が確認できる。この視細胞に分化した細胞をGFPで標識することができ、その後の遺伝子発現、機能解析を行うことが可能となった。この技術を用いて分化した蛍光タンパク質標識視細胞を比較することによって疾患メカニズムやドラッグスクリーニングを検討することができる。
In this research, several human induced pluripotent stem cells (hiPSCs) were used to investigate the disease mechanism of retinitis pigmentosa (RP). Genomic mutation of RP patient derived hiPSCs were confirmed by genomic PCR and conventional sequencing. Genome of these cell lines (and wild-type control hiPSCs) were edited with CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated proteins). In these cell lines, photoreceptor specific promoter GFP expression cassette were inserted in both alleles of AAVS1 sites, safe harbor for gene insertion, thus derived photoreceptors from these hiPSC lines were fluorescently labeled with GFP. By using these cell lines, the gene expressions and the cellular function could be investigated specifically in photoreceptors. These fluorescently labelled cells could be used for the investigation of disease mechanisms and the drug screening.
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研究種目 : 基盤研究(C)(一般)
研究期間 : 2016～2018
課題番号 : 16K11305
研究分野 : 神経科学
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