利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型

doi:10.16288/j.yczz.20-138

遗传 ›› 2020, Vol. 42 ›› Issue (10): 1017-1027.doi: 10.16288/j.yczz.20-138

利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型

林珉婷^1,², 赖璐璐¹, 赵淼¹, 林必玮¹, 姚香平^1,³()

^1. 福建医科大学附属第一医院神经内科,福州 300005
^2. 福建医科大学神经科学研究院,福州 300004
^3. 福建省分子神经病学重点实验室,福州 300005

收稿日期:2020-05-18 修回日期:2020-08-18 出版日期:2020-10-20 发布日期:2020-10-12
通讯作者: 姚香平 E-mail:119373522@qq.com
作者简介:林珉婷,本科,助理研究员,研究方向：神经系统遗传病。E-mail: mintinglin@fjmu.edu.cn
基金资助:
国家自然科学基金青年科学基金项目编号(81801129);福建医科大学启航基金项目资助编号(2017XQ1071);福建医科大学启航基金项目资助编号(2018QH2035)

Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system

Minting Lin^1,², Lulu Lai¹, Miao Zhao¹, Biwei Lin¹, Xiangping Yao^1,³()

^1. Department of Neurology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China
^2. Institute of Neurosciences, Fujian Medical University, Fuzhou 350004, China
^3. Fujian Key Laboratory of Molecular Neurology, Fuzhou 350005, China

Received:2020-05-18 Revised:2020-08-18 Online:2020-10-20 Published:2020-10-12
Contact: Yao Xiangping E-mail:119373522@qq.com
Supported by:
Supported by the National Natural Science Foundation of China No)(81801129);Startup Fund for Scientific Research of Fujian Medical University Nos(2017XQ1071);Startup Fund for Scientific Research of Fujian Medical University Nos(2018QH2035)

摘要/Abstract

摘要：

原发性家族性脑钙化症(primary familial brain calcification, PFBC)是慢性进展性的神经系统遗传病,临床症状主要包括运动障碍、认知障碍及精神障碍等,其致病机制尚未完全明确。研究表明SLC20A2是该病最主要的致病基因。由于Slc20a2基因全身性敲除小鼠模型会导致胎儿生长受限,为更好地研究PFBC发病机制,本研究应用CRISPR/Cas9技术构建了纹状体Slc20a2基因条件性敲除小鼠模型。首先,针对Slc20a2基因编码区,设计3条靶向exon3的sgRNA (single guide RNA),通过构建质粒、转染细胞、Surveyor assay等实验验证sgRNA的活性。其次,选取活性较高的sgRNA重组包装AAV-Cre病毒,应用立体定位将AAV病毒定点注射于小鼠纹状体。体外实验结果表明设计的3条sgRNA均能够有效地介导Cas9切割靶DNA。细胞免疫荧光实验结果证实AAV-Cre病毒具有Cre重组酶活性。最后,通过小鼠脑部组织免疫组化、TA-克隆、高通量测序及Western blot方法检测Slc20a2基因敲除效率,发现实验组小鼠纹状体组织Slc20a2表达明显降低。本研究成功设计了3条能够敲除Slc20a2的功能sgRNA,并应用CRISPR/Cas9技术成功构建了纹状体Slc20a2基因条件性敲除小鼠,为研究PFBC的发病机制提供了有效的动物模型。

关键词: CRISPR/Cas9, Slc20a2, sgRNA, 基因敲除, 小鼠模型

Abstract:

Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.

Key words: CRISPR/Cas9, Slc20a2, sgRNA, gene knockout, mice model

林珉婷, 赖璐璐, 赵淼, 林必玮, 姚香平. 利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型[J]. 遗传, 2020, 42(10): 1017-1027.

Minting Lin, Lulu Lai, Miao Zhao, Biwei Lin, Xiangping Yao. Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system[J]. Hereditas(Beijing), 2020, 42(10): 1017-1027.

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引物名称	碱基序列(5?→3?)
sgRNA1	F:caccGCTCGTGGCGATTGGCCCGAA
sgRNA1	R:aaacTTCGGGCCAATCGCCACGAGC
sgRNA2	F:caccGGCTTCTCACTCGTGGCGAT
sgRNA2	R:aaacATCGCCACGAGTGAGAAGCC
sgRNA3	F:caccGTCGGGGACTCACTGCATCGT
sgRNA3	R:aaacACGATGCAGTGAGTCCCCGAC

利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型

Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system

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