基于Cre/LoxP系统建立睾丸Occludin基因敲除小鼠模型

Establishment of a mouse model of testicular Occludin gene knockout based on the CRE/LOXP system

目的:基于Cre/LoxP系统建立睾丸Occludin基因敲除小鼠模型,并进一步观察其表型变化差异。方法:使用Cre-LoxP系统构建Occludin-floxed(基因型Floxp/-)小鼠,与AQP2-cre(基因型Cre/-)小鼠进行杂交,得到睾丸内Occludin条件性敲除小鼠(基因型Floxp/Floxp Cre/-。利用PCR及Southern印迹技术鉴定F1代敲除小鼠基因型,利用qPCR,Western印迹以及免疫组化鉴定Occludin敲除小鼠中Occludin蛋白表达来验证Occludin敲除小鼠获得成功。比较实验鼠与正常鼠的精子数量,分析其生育能力。结果:Southern印迹和PCR结果我们成功地获得了Occludin基因敲除的目标小鼠。Occludin基因敲除小鼠较正常小鼠Occludin蛋白表达减少,精子数量减少,生育能力降低。差异显著(P<0.05)。结论:采用Cre-LoxP技术可成功构建Occludin基因敲除小鼠,并且Occludin的缺乏会对小鼠的生殖功能产生影响。

Objective:To establish a testicular Occludin gene knockout model in mice and observe the phenotypic changes.Methods:Occludin-floxed(genotype Floxp/-)mice were constructed based on the Cre/loxp system,which were cross-bred with AQP2-cre(genotype Cre/-)mice to derive Occludin knockout mice(Genotype Floxp/Floxp Cre/-).The genotype of the F1 knockout mice was identified by PCR and Southern blot technology.The expression of the Occludin protein in the knockout mice was determined by qPCR,Western blot and immunohistochemistry to verify the success of the modeling.Comparisons were made in the sperm count between the model and normal mice,followed by analysis of their fertility.Results:The target mice of Occludin knockout were successfully constructed,which,compared with the normal controls,showed significantly down-regulated expression of the occludin protein,decreased sperm count and reduced fertility(P<0.05).Conclusion:Occludin gene knockout mice were successfully constructed,and deletion of Occludin affects the reproductive function of the mice.