SNX9基因敲除小鼠模型的构建及表型分析

Construction and phenotypic analysis of SNX9 gene knockout mouse model

目的利用CRISPR/Cas9技术构建分选连接蛋白9(sorting nexin 9,SNX9)基因敲除小鼠模型并对其表型进行分析。方法针对C57BL/6小鼠SNX9基因第3~5外显子,设计gRNA靶位点并在体外转录获得sgRNA,与编码Cas9的mRNA混合后通过受精卵显微注射方法获得F0代阳性敲除小鼠。通过繁育及基因型鉴定获得SNX9基因敲除纯合子小鼠(SNX9^(-/-)小鼠);取小鼠尾部组织提取DNA,用PCR和琼脂糖凝胶电泳进行基因型鉴定;提取SNX9基因敲除小鼠脑组织蛋白,用Western blot检测SNX9蛋白和小胶质细胞标志蛋白的表达水平;记录SNX9基因敲除小鼠的繁殖率和体重,并与野生型小鼠比较;步态系统分析SNX9基因敲除小鼠的步态情况;苏木精-伊红(HE)染色观察小鼠脑组织的形态结构。结果F1代杂合子(SNX9^(+/-))小鼠繁育后可获得3种基因型:野生型(SNX9^(+/+))、杂合子(SNX9^(+/-))、纯合子(SNX9^(-/-));鼠尾提取的DNA用聚合酶链反应(PCR)能鉴定出小鼠的基因型,SNX9敲除小鼠脑组织的SNX9蛋白表达显著低于野生型小鼠;SNX9基因敲除小鼠可正常生长繁殖,但繁殖率显著低于野生型小鼠(P<0.001),体重无显著差异(P>0.05);SNX9基因敲除小鼠脑组织形态学特征与野生型小鼠相比无明显差异;SNX9基因敲除小鼠脑组织的小胶质细胞标志蛋白表达水平与野生型小鼠相比差异无统计学意义(P>0.05)。结论利用CRISPR/Cas9技术成功获得SNX9基因敲除小鼠,为研究SNX9基因的生物学功能和调控机制提供了实验动物模型。

Objective To construct sorting nexin 9(SNX9)gene knockout mouse model using CRISPR/Cas9 technology and analyze its phenotype.Methods sgRNA target sites were designed for exon 3~5 of SNX9 gene,and gRNA was transcribed in vitro.After mixing with mRNA encoding Cas9,F0 generation positive knockout mice were obtained by microinjection of fertilized eggs.SNX9 gene knockout homozygous(SNX9^(-/-))mice were obtained by breeding and genotype identification.DNA was extracted from the tail tis-sue of mice,and genotypes were determined by polymerase chain reaction(PCR)and agarose gel electrophoresis.The expressional lev-els of SNX9 and microglia marker proteins were detected by Western blot in SNX9 gene knockout mice.The reproductive rate and body weight of the SNX9 gene knockout mice were recorded.The gait of SNX9 gene knockout mice was analyzed by gait system.Hematoxylin-eosin(HE)staining was used to observe the brain structure of mouse.Results F1 heterozygous(SNX9^(+/-))mice mated to obtain three genotypes:wild type(SNX9^(+/+)),heterozygous(SNX9^(+/-)),and homozygous(SNX9^(-/-)).DNA derived from mouse tail was used to identify mouse genotypes by PCR,and the level of SNX9 protein of SNX9 knockout mice was significantly lower than that of the wild-type mice.SNX9 gene knockout mice could grow and reproduce normally,but its reproduction rate was lower than that of wild type mice,with normal body weight index.There were no significant differences(P>0.05)in the brain morphology and expressions of microglial marker proteins between SNX9 gene knockout mice and wild-type mice.Conclusion SNX9 gene knockout mouse model was successfully constructed using CRISPR/Cas9 techniques,which provided an experimental animal model for studying the biological func-tion and regulatory mechanism of SNX9 gene.