遺伝子増幅の分子機構には未解明の部分が多く、そのため十分な制御が出来ず、偶然と選択に頼らざるを得ない状況が続いている。そこで我々は、出芽酵母をモデルに、制御可能な特定の分子機構に基づき、しかも選択圧を必要としない新規遺伝子増幅法BiTRExを開発した。BiTRExは、Cas9ニッケースで導入したニックによって縦列反復の隣接部位で進行中の複製フォークを崩壊させて単一末端からなるDNA 2本鎖切断を惹起し、それに対する修復機構break-induced replicationが縦列反復構造によって高い頻度で非アレリックに作動することに基づく。我々は、BiTRExによって16コピーのCUP1アレイ (約32 kb) を~500コピー (~1 Mb) にまで伸長させることに成功した。また、CUP1アレイのみならず、ENA1アレイやCUP1遺伝子座に挿入した~10 kbの反復単位からなる人工遺伝子アレイも伸長できたことから、BiTRExが合成ゲノミクスの様々な応用を可能にする汎用性のある手法と考えられた。当日は、上記研究結果を基に構築した、簡易的に複数遺伝子を増幅させる手法を紹介し、BiTRExによる遺伝子増幅の種々の研究への応用可能性を提示したい。

受賞区分：国内学会・会議・シンポジウム等の賞  受賞国：日本国

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell Genomics  

Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here, we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has just replicated the array, forming a single-ended double-strand break. This break is subsequently end-resected to become single stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching ∼1 Mb. Furthermore, appropriate splint DNAs allow BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.

DOI： 10.1016/j.xgen.2025.100811

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PubMed

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： https://doi.org/10.17912/micropub.biology.001582

リポジトリ公開URL： https://hdl.handle.net/2324/7358365

記述言語：英語  

The lung airways are characterized by long and wide proximal branches and short and thin distal branches. In this study, we investigated the emergence of this hierarchical structure through experimental observations and computational models. We focused on the branch formation in the pseudoglandular stage and examined the response of mouse lung epithelium to fibroblast growth factor 10 (FGF10) by monitoring extracellular signal-regulated kinase (ERK) activity. The ERK activity increased depending on the epithelial tissue curvature. This curvature-dependent response decreased as the development progressed. Therefore, to understand how these epithelial changes affect branching morphology, we constructed a computational model of curvature-dependent epithelial growth. We demonstrated that branch length was controlled by the curvature dependence of growth that was consistent with the experimental observations and lung morphology. However, the branching of the thin branches is suppressed in this model, which is inconsistent with the fact that thin branches in the lung are short. Thus, we introduced branch formation by apical constriction, which was shown to be regulated by Wnt signaling in our previous studies. Mathematical analysis indicated that the effect of apical constriction is cell shape-dependent, suggesting that apical constriction ameliorates the branching of thin branches. Finally, we were able to provide clarity on the hierarchical branching structure through an integrated computational model of curvature-dependent growth and cell shape regulation. We proposed that curvature-dependent growth involving FGF and Wnt-mediated cell shape regulation coordinate to control the spatial scale and frequency of branch formation.

DOI： 10.1101/2023.03.23.533381

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41467-023-44083-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： https://doi.org/10.1038/s41598-021-87912-6

開催年月日： 2023年12月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神戸ポートアイランド   国名：日本国  

開催年月日： 2025年3月

記述言語：英語   会議種別：ポスター発表  

国名：日本国  

開催年月日： 2024年11月

記述言語：日本語   会議種別：ポスター発表  

開催地：福岡国際会議場、マリンメッセA館・B館   国名：日本国  

開催年月日： 2024年10月 - 2024年11月

記述言語：英語  

開催年月日： 2024年10月 - 2024年11月

記述言語：英語  

記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

Introduction: Drug transporters are expressed in a number of tissues such as the intestine, liver, and kidney, and play key roles in drug absorption, distribution, and excretion. Variations in drug transporter gene expression significantly contribute to interindividual differences in drug responses. Epigenetic regulation of drug transporter genes has recently emerged as an important mechanism. Epigenetic regulation alters the expression of genes without changing DNA sequences. Epigenetic control mechanisms are associated with DNA methylation, histone modifications, and microRNAs. Herein we discuss recent advances in the study of the transcriptional and post-transcriptional mechanisms of drug transporters with a focus on epigenetic regulation. Areas covered: This review summarizes recent research on the epigenetic regulation of drug transporter genes, and highlights the importance of identifying novel biomarkers based on epigenetics for use in individualized drug therapy. Expert opinion: Researchers are actively attempting to elucidate the epigenetic mechanisms that control the expression of drug transporters, which affect the pharmacokinetics of drugs. Current evidence suggests that epigenetic changes play an important role in drug transporter function. A clearer understanding of epigenetic regulation in drug transporter genes will provide an insight into novel approaches to individualized drug therapy.

DOI： https://doi.org/10.1080/17425255.2017.1230199

種別：学会・研究会等 

種別：学会・研究会等 

種別：大会・シンポジウム等 

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執筆者：本人