利用CRISPR/Cas9技术将LoxP序列靶向引入小鼠Alk1基因  

Targeted Insertion of Lox P Sequences into Alk1 Gene in Mice by Using CRISPR/Cas9

作　　者：徐铭[1] 徐宏治[2] 陈峻叡 秦智勇[2] 陈衔城[2] 

机构地区：[1]复旦大学附属华山医院麻醉科,200040 [2]复旦大学附属华山医院神经外科,200040

出　　处：《中国临床神经科学》2015年第6期615-623,共9页Chinese Journal of Clinical Neurosciences

基　　金：上海市科学技术委员会实验动物研究领域项目(编号:13140903300);国家自然科学基金(编号:81000489)

摘　　要：目的将LoxP序列靶向引入小鼠4腩1基因，拟通过Cre／LoxP系统条件性敲除4腩1基因建立脑动静脉畸形小鼠模型。方法利用CRISPR／Cas9技术编辑小鼠Alkl基因：①设计和选择两个单导向RNA（sgRNA）分别识别外显子3—4和8～9的非编码区位点；②设计带有2个LoxP序列且与靶基因同源的供体载体；⑧将体外合成的sgRNA，Cas9mRNA~I]供体载体注射到小鼠受精卵；④受精卵经假孕小鼠代孕出生后，经PcR和测序筛选目标小鼠。结果小鼠Alkl等位基因的特定位点插入了LoxP序列。结论CRISPR／Cas9技术能成功将LoxP序列靶向引入小鼠Alkl基因，为建立脑动静脉畸形小鼠模型创造条件。Aim To generate mutant mice with Alkl gene inserted by LoxP sequences, by conditional deletion of Alkl via the Cre/LoxP system, to further develop brain arteriovenous malformation （AVM） mouse models. Methods The CRISPR/Cas9 technology was used to edit Alkl. Two single guide RNA （sgRNA） with the recognition sites between exon 3 to 4 and exon 8 to 9 were designed and selected. A donor vector homologous to the target gene with two additional LoxP sequences was designed. In vitro synthesized sgRNA, Cas9 mRNA and donor vector were injected to mouse zygotes, which were transferred to pseudopregnant mice. Mutant mice were identifed by gene analysis. Results The Alkl allele was inserted by LoxP sequences in the targeted sites. Conclusion The CRISPR/Cas9 technology can successfully generate the mutant mice, which is a basis for the creation of brain AVM mouse models.

关 键 词：CRISPR/Cas9 Alk1基因 小鼠 动静脉畸形 

分 类 号：Q189[生物学—神经生物学] R743.4[生物学—普通生物学]