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针对性地将条件表达卡塞特插入小鼠基因组,使用修改后的i-PITT

Hiromi Miura¹, Ayaka Nakamura², Aki Kurosaki¹

¹Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Kanagawa, Japan.

|June 5, 2024

概括

这项研究引入了一种创建转基因小鼠的新方法,允许插入条件表达盒. 这提高了用于研究的转基因小鼠生成的可靠性和效率.

科学领域:

遗传学是一个遗传学.
分子生物学分子生物学
转基因技术转基因技术

背景情况:

通过前核注射的传统转基因 (Tg) 鼠标生成通常会导致由于随机DNA插入而导致不可靠的转基因表达.
基于前核注射的向转基因 (PITT) 和改进的PITT (i-PITT) 已开发,通过将插入指向特定的基因组位置来提高转基因表达的一致性.
由于LoxP位点之间的干扰,PITT的一个局限性是它无法使用Cre-LoxP重组来插入条件表达盒.

研究的目的:

为了克服i-PITT在插入条件表达式磁带方面的局限性.
开发一种经过修改的i-PITT方法,与Cre-LoxP局部特定再组合相兼容,用于条件转基因.

主要方法:

通过同时使用PhiC31整合酶 (PhiC31o) 和FLP复合酶 (FLPo) mRNAs来修改i-PITT方法.
通过在创始老鼠的Rosa26位点创建条件表达卡塞特来演示该策略.

主要成果:

经过修改的i-PITT方法成功地在Rosa26位点插入了条件表达盒,效率为13.7%.
包括FLPo mRNA有效地阻止了向量脊柱在创始老鼠基因组中的插入.

结论:

在i-PITT方法中同时应用PhiC31整合酶和FLP重组酶,可以插入携带基于Cre-LoxP的条件表达盒的供体质粒.
这一进步有助于为生物医学研究生成更可靠,更通用的转基因小鼠模型.

关键词:

条件表达式是一种有条件的表达式.在FLP-FRT之间.鼠标鼠标是一个鼠标.这就是 PhiC31 整合酶的作用.基于核前注射的有针对性的转基因发生.有针对性的转基因.

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