Lin Min Ting, Lai Lu Lu, Zhao Miao, Lin Bi Wei, Yao Xiang Ping
Department of Neurology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China.
Institute of Neurosciences, Fujian Medical University, Fuzhou 350004, China.
Yi Chuan. 2020 Oct 20;42(10):1017-1027. doi: 10.16288/j.yczz.20-138.
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.
原发性家族性脑钙化（PFBC）是一种慢性进行性神经遗传性疾病。其临床症状主要包括运动障碍、认知障碍和精神损害；发病机制尚不清楚。研究表明，SLC20A2是该疾病最常见的致病基因。由于Slc20a2基因敲除小鼠模型会导致胎儿生长受限，为了更好地了解PFBC的发病机制，本研究利用CRISPR/Cas9技术构建了小鼠纹状体中Slc20a2基因的条件性敲除模型。首先，设计了3条sgRNA（单向导RNA）靶向Slc20a2基因的外显子3。通过构建表达质粒、转染细胞和Surveyor检测验证了各sgRNA的活性。其次，选择活性最高的SgRNA制备重组AAV-Cre病毒，通过立体定位法将其注射到小鼠纹状体中。体外实验表明，3条sgRNA均可有效介导Cas9对各自靶DNA的切割。通过免疫荧光检测证实了AAV-Cre的Cre重组酶活性。采用免疫组化、TA克隆、高通量测序和蛋白质免疫印迹法检测和评估Slc20a2基因敲除效率。结果显示，实验组小鼠纹状体中Slc20a2的表达显著降低。本研究成功设计了3条能够敲除Slc20a2的sgRNA，并利用CRISPR/Cas9技术成功建立了小鼠纹状体中Slc20a2基因的条件性敲除，从而为研究PFBC的发病机制提供了有效的动物模型。
