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Objective To breed and identify the Alg13 knockout (KO) mice to provide an ideal animal model for exploring the role of Alg13 in epilepsy. Methods Alg13 KO and wildtype (WT) mice with C57BL/6J background were used, including 4-week-old Alg13 KO mice (n=12; 6 males and 6 females; 6~7 g), 4-week-old WT mice (n=12; 6 males and 6 females; 7~8 g), 8-week-old Alg13 KO male mice (n=18; 21~23 g) and 8-week-old WT male mice (n=18; 22~24 g). The genotype of Alg13 KO mice was identified using PCR, gel electrophoresis, DNA sequencing, TA cloning and blue-white selection method. Immunohistochemical analysis and Western blotting were applied to verify the deletion of ALG13 protein in Alg13 KO mice. The behavioral manifestations and characteristics of seizures in Alg13 KO mice were evaluated using a synchronized video EEG system. The expression level of voltage-gated sodium channel alpha subunit 1 (SCN1A) protein was measured by Western blot assay. Results The genotyping methods including the gel electrophoresis, DNA sequencing and blue-white selection confirmed a 5-base deletion in Alg13 KO mice. Compared with WT mice, Alg13 KO mice showed a significantly lower ALG13 expression level in the cortical, cerebellar and hippocampal regions in the immunohistochemical (P < 0.001, P < 0.05, P < 0.001) and Western blot assay (P < 0.001). Besides, some Alg13 KO mice developed frequent spontaneous seizures with typical behavioral manifestations and EEG changes of the epileptic seizure. Alg13 KO mice had significantly higher δ and θ wave energy value of EEG compared with the WT group (P < 0.001). The expression levels of SCN1A in the hippocampus and cortex were remarkably higher in Alg13 KO mice than those in WT mice (P < 0.05, P < 0.01). Conclusion The Alg13 knockout mouse model can be obtained successfully, which shows significant epilepsy susceptibility and lays a foundation for studying the mechanism of the epilepsy caused by genetic abnormalities.