Construction Of Ext1^+/- Knockout Mouse Model Using CRISPR/Cas9 Technology

Objective:Hereditary Multiple Exostoses（HME）is an autosomal dominant skeletal disorder,which often leads to short stature and bone deformity.Its main pathogenic gene is EXT1,but the pathogenesis and mechanism of HME are still unclear.In this study,an Ext1^+/-gene knockout mouse model was constructed based on CRISPR/Cas9 technology to explore more biological functions of the EXT1 gene,and to provide a theoretical rationale for biomedical research such as studying the pathogenesis of HME and discovering new drug targets.Methods:Two sg RNAs were designed to target mouse Ext1 gene.sg RNA and Cas9 protein were injected into the cytoplasm of fertilized eggs at prokaryotic stage.F0 generation Ext1 gene knockout mice were obtained after embryo transfer.After propagation,the phenotype of F2 generation Ext1^+/-gene heterozygous mutant mice was identified.The molecular expression level of Ext1 gene in gene knockout mice was detected by q PCR,WB and IHC experiments.X-ray was used to analyze the morphological characteristics of the bone of Ext1^+/-gene knockout mice,and the development of articular cartilage was detected by pathological section of knee joint.In the same time,body weights and lengths were recorded in Ext1^+/-gene knockout mice and wild-type mice.Results:We obtained two F0 generation Ext1 knockout mice,and obtained a mouse strain with a heterozygous deletion of 46 bp in Ext1 gene and stable inheritance after breeding.The expression level of Ext1m RNA in Ext1^+/-gene knockout mice was significantly lower than that in wild-type mice,but there was no significant statistical difference in protein expression level in liver and knee joint between Ext1^+/-gene knockout mice and wild-type mice.However,IHC experiments showed that the content of EXT1 protein in knee cartilage cells of Ext1^+/-knockout mice was lower than that of wild-type mice.HE staining showed that the morphology and structure of heart,liver,lung,kidney and knee-joint of Ext1^+/-knockout mice were normal.X-ray analysis showed that 14 male Ext1^+/-knockout mice had abnormal development near the caudal vertebra,and the CT value was about 200Hu.In addition,the body weight of Ext1^+/-knockout mice was lower than that of wild-type mice,and there was no significant difference in body length.Conclusion:We successfully constructed the Ext1^+/-knockout mouse model,and the weight of Ext1^+/-knockout mice was slightly lower than that of wild-type mice.No osteochondroma was found in the bones of mice,but there was male sex-dependent abnormal development in Ext1^+/-knockout mice,and its CT value was between the rib and spine of mice,indicating abnormal bone development.