generation of HLA homogenized iPS cell bank using induced homologous recombination method
| Project/Area Number |
18K19448
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| Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
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| Allocation Type | Multi-year Fund |
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| Review Section |
Medium-sized Section 49:Pathology, infection/immunology, and related fields
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| Research Institution | Osaka University |
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Principal Investigator |
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| Project Period (FY) |
2018-06-29 – 2021-03-31
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| Project Status |
Completed (Fiscal Year 2020)
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| Budget Amount *help |
¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000)
Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2019: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2018: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
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| Keywords | 相同組換え / iPS細胞 / 再生医療 / 免疫拒絶 / ゲノム編集 / 創薬モデル / 染色体交差 / ブルーム遺伝子 / HLA / 病因因子 |
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| Outline of Final Research Achievements |
Here, we developed a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor ML216 exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6, where human leukocyte antigen (HLA) loci are located. Genotyping and flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions.
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| Academic Significance and Societal Importance of the Research Achievements |
我々はゲノム編集技術と、低分子化合物によるブルーム蛋白の一過性に阻害により、染色体上の狙った領域のみで相同組換えを生じさせる技術を開発した。この技術をHLA領域に適用する事により、免疫原性を低減させた移植用iPS細胞の作出に繋げる事が可能であり、この社会的意義は大きいものがあると考えられる。また本技術は、近年の解析技術の向上により割り出されたゲノムワイド関連解析によって見出された病因因子の表現型との相関解析にも役立つ事が期待され高い学術的意義を示すと考えられる。
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Report
(4 results)
Research Products
(7 results)
