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用于DNA甲基化编辑打印位点和影响评估的协议

Yoichi Sekita¹, Tohru Kimura²

¹Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, Sagamihara, Kanagawa, Japan. ysekita@kitasato-u.ac.jp.

Methods in molecular biology (Clifton, N.J.)

|July 16, 2024

概括

这项研究详细介绍了一种使用CRISPR/dCas9和SunTag系统的DNA甲基化编辑协议,用于改变Dlk1-Dio3印记域中的甲基化. 这种方法使研究人员能够研究甲基化.

科学领域:

表观遗传学在表观遗传学中,表观遗传学是指表观遗传学.
分子生物学分子生物学
基因组学就是基因组学.

背景情况:

基因甲基化对于调节基因表达和基因组印记至关重要.
Dlk1-Dio3位点是一个具有良好的特征的印记域,对发育至关重要.
了解动态DNA甲基化变化对于破译表观遗传调节至关重要.

研究的目的:

介绍一种用于向DNA甲基化编辑的新型实验协议.
调查诱导的DNA甲基化变化对Dlk1-Dio3域内印记状态的影响.
提供表观遗传修饰和基因表达的等位基因特异性分析方法.

主要方法:

使用CRISPR/dCas9和SunTag系统进行针对性的DNA甲基化编辑.
引入编码DNA甲基转移酶或TET酶的等离子体载体,以便在ES细胞中暂时表达.
采用基因基因基因甲基化,基因表达和基因组蛋白修饰的基因特异性定量分析.

主要成果:

在Dlk1-Dio3印记域中成功诱导了DNA甲基化的目标增益或损失.
证明了研究甲基化特征影响印记状态,基因表达和基因素修饰的能力.
建立了全面评估位置印记状态的策略.

结论:

提出的协议为研究DNA甲基化动态和印记提供了一个强大的工具.
这种方法可以对整个印记领域的表观遗传调节进行详细的研究.
开发的策略有助于对表观遗传修饰及其功能后果进行深入分析.

关键词:

对等位基因特异性的基因表达分析分析.在CRISPR/dCas9系统中.通过DNA甲基化.基因组印记是指基因组的印记.印制控制区域 (ICR) 是指印制控制区域.在 SunTag 系统中使用 SunTag.有针对性的DNA甲基化编辑.

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