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大规模的全基因组优化和预测Cre重组酶系统用于小鼠精确的基因组操纵

Valerie Erhardt¹, Elli Hartig^1,2, Kristian Lorenzo^1,3

¹The Jackson Laboratory for Mammalian Genetics, Bar Harbor, ME.

bioRxiv : the preprint server for biology

|July 1, 2024

概括

在小鼠中优化Cre-Lox重组对于高效的基因编辑至关重要. 关键因素包括loxP站点距离,突变站点和Cre驱动器年龄,改善条件淘汰赛小鼠的产生.

科学领域:

遗传学和基因组学在
分子生物学分子生物学
转基因动物模型

背景情况:

在老鼠遗传学中,Cre-Lox重组系统对于条件基因操纵至关重要.
挑战包括高成本,时间投资和影响实验结果的不可预测的重组事件.
了解影响Cre介导重组的因素对于优化其应用至关重要.

研究的目的:

系统地分析影响小鼠CRE中介重组效率的全基因组因素.
确定最佳条件,以降低成本和时间生成条件淘汰鼠标模型.
为选择有效的Cre-Lox策略提供基于数据的建议.

主要方法:

在C57BL/6J背景上的ROSA26位点生成了11个具有条件等位基因的新型小鼠菌株.
基于loxP间距离,突变的loxP位点,等位基结,染色体位置和育种者年龄的重组效率的检查.
对CRE驱动菌株选择对重组的影响的系统分析.

主要成果:

通过1-4kb间距离的loxP位点实现最佳重组;突变的loxP位点在1-3kb间有效.
在≥15kb (野生型loxP) 和≥7kb (突变型loxP位点) 时,完全重组失败.
育种者年龄 (8-20周),异合的合基因,和loxP间距离≤4kb显著提高了重组效率.

结论:

克雷驱动菌株选择和育种年龄对于有效的克雷-洛克斯重组至关重要.
特定的loxP间距离和loxP位点类型决定了重组成功.
这项研究提供了优化Cre-Lox技术的指导方针,用于生成精确的小鼠模型,节省时间和资源.

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