Efficient Detection of Flox Mice Using In Vitro Cre Recombination
Efficient Detection of Flox Mice Using In Vitro Cre Recombination
复制标题
利用体外 Cre 重组有效检测 Flox 小鼠
DOI：
10.1007/978-1-0716-3016-7_12
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发表时间：
2023
影响因子：
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通讯作者：
Hatada Izuho
中科院分区：
文献类型：
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作者：
Kobayashi Ryosuke;Horii Takuro;Hatada Izuho
CRISPR/Cas9基因组编辑技术的进步已经允许快速产生Cre-loxP条件性敲除小鼠。然而，目前对flox小鼠进行基因分型的策略（通常基于从数十只幼崽克隆目标序列后的桑格测序）非常耗时。在这里，我们描述了一种快速筛选方法的flox小鼠，使用体外Cre重组，可以使用简单的酶促反应进行，并能够在1天内检测功能的flox小鼠。此外，我们引入了一个有效的策略，随后的序列分析克隆的floxed区域使用的In-Fusion系统。我们的基因分型管道减少了繁重的任务，从而有助于快速选择准确编辑的flox小鼠。
Advances in CRISPR/Cas9 genome editing technologies have allowed for the rapid generation of Cre-loxP conditional knockout mice. However, current strategies for genotyping flox mice, typically based on Sanger sequencing following cloning of target sequences from dozens of pups, are time-consuming. Here, we describe a rapid screening method for flox mice, using in vitro Cre recombination that can be performed using simple enzymatic reactions and enables detection of functional flox mouse within 1 day. In addition, we introduce an efficient strategy for subsequent sequence analysis by cloning of floxed regions using the In-Fusion system. Our genotyping pipeline reduces laborious tasks and thus contributes to the rapid selection of accurately edited flox mice.