Construction of mice model of Zfp212 gene knockout and exploring its effects on female fertility
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摘要:
目的:通过构建基因敲除小鼠模型探究锌指蛋白212(Zinc finger protein 212,Zfp212)对于雌性生育力的影响。方法:利用CRISPR/Cas9技术构建Zfp212基因敲除小鼠;利用实时荧光定量PCR、Western blot、免疫荧光实验检测Zfp212蛋白表达、定位情况,并对Zfp212基因敲除效率进行验证;通过卵巢切片和HE染色、体外受精及早期胚胎培养和生育力测试实验对Zfp212基因敲除雌性小鼠的生殖表型进行分析。结果:Zfp212基因在卵母细胞和早期胚胎中高表达,且呈母源表达模式;基因敲除效率验证实验结果显示Zfp212敲除小鼠构建成功;Zfp212基因敲除雌性小鼠的卵泡发育、卵母细胞成熟、受精、植入前胚胎发育以及平均每窝产仔数量与对照组小鼠相比,差异均无统计学意义。结论:Zfp212基因对雌性小鼠的生育力建立不是必需的。
Abstract:
Objective:The study aims to investigate the role of Zfp212 on female fertility by generating Zfp212 knockout mice model. Methods:CRISPR/Cas9 system was used to generate Zfp212 knockout mice. Zfp212 expression pattern,subcellular location and Zfp212 knockout efficiency were detected by quantitative real-time PCR(RT-qPCR),immunofluorescence staining(IF)and Western blot. Ovary histological analysis and HE staining,in vitro fertilization(IVF),in vitro embryo culture and fertility test were performed to explore the effects of Zfp212 knockout on female mice fertility. Results:Zfp212 was expressed dominantly in oocytes and early embryos,and it showed a maternal expression pattern. The results of Western blot and sanger sequencing showed Zfp212 knockout mice model was constructed successfully. Compared with control groups,Zfp212 knockout female mice had no significant differences in follicle development,oocyte maturation,fertilization,preimplantation embryo development and litter size. Conclusion:These results indicated that Zfp212 gene is not necessary for the establishment of female fertility in mice.