CSE基因敲除小鼠的基因型鉴定方法

The genotyping method for CSE knockout mice

目的介绍一种胱硫醚-γ-裂解酶(Cystathionine-γ-lyase,CSE)基因敲除小鼠基因型的鉴定方法。方法提取3只野生型(Wild type,WT)小鼠(CSE^(+/+))和21只杂合子小鼠(CSE^(+/-))繁殖的子代小鼠的鼠尾基因组DNA,以其为模板,聚合酶链反应(Polymerase chain reaction,PCR)扩增后凝胶电泳;然后分别提取3只WT小鼠和6只随机选择的CSE基因敲除小鼠(CSE^(-/-))肠道组织和骨髓来源的巨噬细胞(Bone marrow-derived macrophage,BMDM)蛋白,并用免疫印迹法(Western blot)检测其中CSE蛋白的表达。结果凝胶电泳结果显示:只在167 bp处有表达的是WT鼠或CSE^(+/+)小鼠;在167 bp处、309 bp处均有表达的是CSE^(+/-)小鼠;只在309 bp处有表达的是CSE^(-/-)小鼠。Western blot检测结果表明,与野生鼠相比,CSE^(-/-)小鼠肠道组织和BMDM中CSE蛋白表达显著性降低(P<0.01),与凝胶电泳实验结果一致。结论采用PCR结合凝胶电泳的方法鉴定CSE基因敲除小鼠的基因型是一种较为准确的鉴定方法,此方法可以为使用该动物的实验研究节约经济成本和时间成本。

Objective To introduce a genotyping method for CSE knockout mice.Methods Mouse tail genomic DNA was extracted from wild type(WT)mice(n=3)and offspring mice via heterozygous breeding(n=21),respectively.Then,the target fragments were amplified by polymerase chain reaction(PCR)and verified with gel electrophoresis.Furthermore,the proteins of intestinal tissue and bone marrow-derived macrophage(BMDM)from WT mice(n=3)and CSE^(-/-)mice were extracted(n=6),respectively,and then the CSE protein expression was detected by western blot.Results The results of gel electrophoresis showed that 167 bp target fragment was verified in both WT mice and CSE^(+/+)mice,however,both 167 bp and 309 bp target fragment were verified in CSE^(+/-)mice,and only 309 bp target fragment was verified in CSE^(-/-)mice.Western blot data showed that the CSE protein expression from intestinal tissue and BMDM of CSE^(-/-)mice was significantly decreased compared with those of WT mice(P<0.01),which was consistent with the results of gel electrophoresis.Conclusion PCR combined with gel electrophoresis might be a relatively accurate method to identify the genotype of CSE knockout mice,which may be economical and time saving method for the experimental study using CSE knockout mice.