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在Msh2淘汰HC11细胞中,Rad51和DNA结合酶IV核蛋白表达的减少诱导了在β-casein基因位点的CRISPR/Cas9介导的低淘汰效率

Ga-Yeon Kim¹, Man-Jong Kang¹

¹Department of Animal Science, Chonnam National University, Gwangju 61186, Korea.

Animal bioscience

|November 1, 2024

概括

击败Msh2 (不匹配修复基因) 通过降低Rad51和DNA结合酶IV的调节,降低同源重组 (HR) 效率. 这影响了小鼠β-casein locus的基因编辑成功.

科学领域:

分子生物学分子生物学
基因编辑基因编辑
修复DNA的途径

背景情况:

同源重组 (HR) 效率对于成功的基因敲进系统至关重要.
涉及Msh2的DNA不匹配修复 (MMR) 途径与HR密切相关.
Msh2 识别了 HR 期间的不匹配,从而影响了其效率.

研究的目的:

调查Msh2淘汰对HR介导的淘汰效率的影响.
分析Msh2抑制对Rad51和DNA结合酶IV表达的作用.
探索Msh2在小鼠β-素基因位点内置中所扮演的角色.

主要方法:

HC11细胞与敲入载体,导向RNA和Msh2siRNA的共同感染.
使用聚合酶链反应 (PCR) 评估敲门效率.
通过RT-qPCR和Western blot对Msh2,Rad51和DNA结合酶IV表达的评估.

主要成果:

在HC11细胞中,Msh2 knockdown显著降低了 knock-in 矢量效率.
低调Msh2蛋白导致Rad51和DNA结合酶IV的核表达减少.
观察到与HR相关的Rad51和与NHEJ相关的DNA结合酶IV显著下降.

结论:

减少Msh2蛋白表达会降低Rad51和DNA结合酶IV的调节.
减少Rad51表达是Msh2淘汰的直接结果.
这导致了人力资源中介的投入效率下降.

关键词:

集群定期间隔的短时间的Palindromic重复/CRISPR相关的9 (CRISPR/Cas9)修复DNA不匹配 (MMR) 的方法同源重组 (HR) 是一种同源的重组.非同类末端连接 (NHEJ)

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