哺乳动物核调节元素的转录合变化
Bo Gu1, Tomek Swigut1, Andrew Spencley1,2
1Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
概括
我们开发了gRNA寡核酸组合 (CARGO) 用于多重导向RNA传递. 这种方法表明,随着活细胞的转录激活,调节元件的移动性会增加.
科学领域:
- 分子生物学
- 遗传学
- 细胞生物学
背景情况:
- 导向RNA (gRNA) 的多重传递对于复杂的基因工程至关重要.
- 了解细胞分化过程中的 cis 调节元件的动态是必不可少的.
研究的目的:
- 开发一种用于将多个不同的gRNA输入单细胞的新方法.
- 在活胚胎干细胞中量化测量增强剂和促进剂的动态.
- 调查 cis 调节元件的移动性与转录活性之间的关系.
主要方法:
- 开发用于多重化gRNA输送的gRNA寡核酸组合 (CARGO) 战略.
- 将CARGO与催化死Cas9 (dCas9) 成像用于基因组位点的活细胞追踪.
- 在分化和对干扰的反应中增强剂和促进剂运动的定量分析.
主要成果:
- 它可以有效地将几十种不同的gRNA输入单个细胞.
- 所有检查的功能元素 (增强剂,促进剂) 都表现出亚扩散性行为.
- 转录激活增加与cis调节元件的移动性增加相关.
- 对RNA聚合酶II活性的干扰逆转了与活性相关的局部移动性的增加.
结论:
- CARGO是多重化gRNA传递和研究基因调节的有效工具.
- 细胞调节元件的移动性与转录活性密切相关.
- 活细胞成像为不同细胞状态中的调节元件的功能提供了动态洞察力.
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