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| 应用CRISPR/Cas9基因编辑技术建立miRNA-301a敲除小鼠 |
| 刘宣1,季青1,李文2,周利红1,王璇2,江海丽1,陈静1,畅立圣1,李琦1* |
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(1. 上海中医药大学附属曙光医院肿瘤科, 上海 201203;
2. 上海中医药大学附属上海市中医医院肿瘤科, 上海 200071
*通信作者) |
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| 摘要: |
| 目的 利用CRISPR/Cas9基因编辑技术建立miRNA敲除小鼠模型。方法 根据miRNA基因序列,设计针对miR-301a的gRNA引物序列 (双靶点),PCR扩增获得针对miR-301a的体外转录模板DNA和构建Cas9体外转录模板,然后进行Cas9和gRNA体外转录。利用体外转录的gRNA/Cas9 mRNA对小鼠受精卵显微注射,并利用T7E1酶切和基因测序对miR-301a突变进行检测和鉴定。结果 PCR扩增、凝胶电泳和基因测序鉴定证实,获得序列正确的用于体外转录的模板DNA;体外转录获得gRNA/Cas9 mRNA,顺利完成对小鼠受精卵的显微注射;利用T7E1酶切对miR-301a突变进行检测,发现8只新生小鼠中有7只(87.5%)被鉴定在miR-301a位点携带突变;基因测序结果显示,各小鼠均有不同程度的碱基插入或缺失突变,其中缺失碱基数目最多的4号小鼠产生31个碱基缺失。结论 成功建立miR-301a基因高效敲除小鼠模型。 |
| 关键词: CRISPR/Cas9 基因编辑 miR-301a 基因敲除小鼠 |
| DOI：10.3724/SP.J.1008.2015.00256 |
| 投稿时间：2014-12-04修订日期：2015-01-29 |
| 基金项目:国家自然科学基金 (81273958, 81303102, 81473478, 81303103),上海市科委科技发展基金实验动物专项基金 (13140902500,14140901402),上海市优秀学科带头人计划 (XBR2011061),上海市教委创新项目 (12ZZ118,13YZ045),上海市教育发展基金会晨光计划 (13CG47),上海市卫生局科研基金 (20114Y001). |
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| Application of CRISPR/Cas9 gene targeting technology for establishing miRNA-301a knockout mouse model |
| LIU Xuan1,JI Qing1,LI Wen2,ZHOU Li-hong1,WANG Xuan2,JIANG Hai-li1,CHEN Jing1,CHANG Li-sheng1,LI Qi1* |
(1. Department of Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
2. Department of Oncology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China
*Corresponding author) |
| Abstract: |
| Objective To establish miRNA knockout mouse model using CRISPR/Cas9 gene targeting technology. Methods According to the gene sequence of miRNA, we designed the primers of the gRNA targeting miR-301a (two targets) and obtained DNA template for in vitro transcription using PCR amplification; then we constructed Cas9 template for in vitro transcription, followed by in vitro transcription for gRNA of Cas9. In vitro transcribed gRNA/Cas9 mRNA was microinjected into the mouse zygote. T7E1 digestion and gene sequencing were used to detect and characterize the mutation of miRNA. Results PCR amplification, gel electrophoresis and gene sequencing proved that we had obtained the correct DNA template targeting miR-301a for in vitro transcription. By in vitro transcription we obtained gRNA/Cas9 mRNA, which was successfully microinjected into mouse zygote. The miR-301a mutants were detected by digestion with T7E1, and it was found that 7 of the 8 (87.5%) neonatal mice were found carrying mutations in miR-301a sites. Gene sequencing results showed that all mice had different levels of nucleotide insertion or deletion mutation, with the maximum number of 31 bases deletion found in No.4 mouse. Conclusion We have successfully established a mouse model with miR-301a gene knocked out, which lays a solid foundation for related future research. |
| Key words: CRISPR/Cas9 gene editing miR-301a knockout mice |
