Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system
Received date: 2020-05-18
Revised date: 2020-08-18
Online published: 2020-10-12
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)
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
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 . DOI: 10.16288/j.yczz.20-138
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