使用基于矢量的CRISPR/Cas9系统在鱼细胞系中移除进化保守的功能MYC域
Chanhee Kim1, Avner Cnaani2, Dietmar Kültz3
1Department of Animal Sciences, University of California, Davis, CA, 95616, USA.
Scientific reports
|July 26, 2023
概括
MYC转录因子对于细胞功能至关重要. 这项研究确立了MYC基因破坏和莫桑比克鱼细胞度调节之间的直接联系,创造了一个新的研究工具.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 细胞生理学 细胞生理学
背景情况:
- MYC转录因子对于跨物种的细胞功能保存至关重要.
- 在动物透调节中MYC的确切作用在机理上仍然不清楚.
- 莫桑比克鱼 (Oreochromis mossambicus) 是一个有价值的模型,因为它的欧性和耐压力.
研究的目的:
- 在莫桑比克鱼脑细胞系中功能性地破坏MYC基因 (myca).
- 建立MYC功能和细胞透调节之间的因果关系.
- 开发一种新的遗传工具,用于研究MYC在细胞生物学中的作用.
主要方法:
- 在O. mossambicus细胞中利用了基于矢量优化的CRISPR/Cas9系统进行基因编辑.
- 采用细胞隔离和稀释策略来产生多克隆和单克隆MYC淘汰 (ko) 细胞系.
- 验证了基因编辑效率,并表征了myca基因中导致的框架转移突变.
主要成果:
- 在鱼细胞中实现了MYC淘汰的高基因编辑效率 (高达98.2%).
- 产生了可行的多克隆和单克隆的myca ko细胞系,具有不同的形态.
- 框架转移突变功能性地使MYC的转录调节和DNA结合域失活.
结论:
- 成功创建了基因改造的鱼细胞系,破坏了MYC功能.
- 这些MYC淘汰细胞系为研究MYC在细胞透调节中的作用提供了一个强大的新工具.
- 这项研究弥合了对MYC在动物透调节中的机理性参与的理解上的差距.
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