CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre-loxP-mediated lineage tracing
CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre-loxP-mediated lineage tracing
复制标题
CRISPR/Cas9 介导的 Rosa26 位点靶向产生 Cre 报告大鼠品系，用于监测 Cre-loxP 介导的谱系追踪
DOI：
10.1111/febs.14188
复制
发表时间：
2017-10-01
期刊：
影响因子：
5.4
通讯作者：
Zhang, Lianfeng
中科院分区：
文献类型：
--
作者：
Ma, Yuanwu;Yu, Lei;Zhang, Lianfeng
大鼠是生理学、毒理学和药理研究的重要实验动物。成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关9（Cas9）是一种简单有效的工具，可在大鼠体内产生精确的基因修饰，这将促进遗传资源的积累，并使得基因功能的研究更加精确。为了监测 Cre-loxP 介导的体内切除，我们通过使用 CRISPR/Cas9 在 Rosa26 位点敲入 Cre 报告基因盒，生成了 Cre 报告基因大鼠品系 (Rosa26-imCherry)。 Rosa26-imCherry 大鼠使用 Cre-loxP 系统表现出 mCherry 盒 (imCherry) 的诱导表达，而正常大鼠则表现出全身普遍存在的 eGFP 表达，但没有 mCherry 表达。将腺相关病毒血清型 9-Cre 注射到海马和骨骼肌中，导致病毒感染细胞中 mCherry 表达。然后通过将 Rosa26-imCherry 大鼠与普遍表达 CAG-Cre 的转基因大鼠、心脏特异性 α-MHC-Cre 转基因大鼠和肝脏特异性 Alb-Cre 敲入大鼠杂交来评估 Cre-loxP 介导的 mCherry 表达。最后，利用所建立的系统追踪了两个内源基因启动子（Wfs1-Cre 敲入大鼠、FabP2-Cre 敲入大鼠）驱动的 Cre 表达模式。总之，我们证明了在 Cre 重组酶存在的情况下通过激活 mCherry 表达来切除 Rosa26-imCherry 大鼠中的 loxP 侧翼等位基因。这种新建立的 Rosa26-imCherry 大鼠品系是促进 Cre 表达模式测定和追踪实验的有用工具。
The rat is an important laboratory animal for physiological, toxicological and pharmacological studies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) is a simple and efficient tool to generate precise genetic modifications in rats, which will promote the accumulation of genetic resources and enable more precise studies of gene function. To monitor Cre-loxP-mediated excision in vivo, we generated a Cre reporter rat strain (Rosa26-imCherry) by knockin of a Cre reporter cassette at the Rosa26 locus using CRISPR/Cas9. Rosa26-imCherry rats exhibited inducible expression of the mCherry cassette (imCherry) using the Cre-loxP system, whereas normal rats exhibited ubiquitous expression of eGFP but not mCherry in the whole body. Injection of adeno-associated virus serotype 9-Cre into the hippocampus and skeletal muscle resulted in mCherry expression in virus-infected cells. Cre-loxP-mediated mCherry expression was then evaluated by crossing Rosa26-imCherry rats with transgenic rats ubiquitously expressing CAG-Cre, heart-specific alpha-MHC-Cre transgenic rats and liver-specific Alb-Cre knockin rats. Finally, using the established system the expression pattern of Cre driven by two endogenous gene promoters (Wfs1-Cre knockin rat, FabP2-Cre knockin rat) was traced. In summary, we demonstrated excision of the loxP-flanked allele in Rosa26-imCherry rats via activation of mCherry expression in the presence of Cre recombinase. This newly established Rosa26-imCherry rat strain represents a useful tool to facilitate Cre-expression pattern determination and tracing experiments.