Laboratory Animal Science ›› 2024, Vol. 41 ›› Issue (6): 27-33.DOI: 10. 3969 / j. issn. 1006-6179. 2024. 06. 006

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Construction and Phenotypic Analysis of Mutant Mice with Partial Deletion of MIWI C-terminal and 3’UTR

  

  1. ( State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing 210029, China)
  • Received:2024-02-23 Online:2024-12-28 Published:2025-01-15

MIWI 蛋白 C-端及 3’UTR 部分缺失小鼠的构建及其表型分析

  

  1. (南京医科大学生殖医学与子代健康全国重点实验室,南京 210029)
  • 通讯作者: 赵婷婷( 1988—) ,女,教师,研究方向为配子发生的分子机制,E-mail:zhaotiti@ njmu. edu. cn。 徐宇君( 1965—) ,男,教授,博士生导师,研究方向为配子发生的分子机制,E-mail: xuyujun@ njmu. edu. cn。
  • 作者简介:鲁 悦( 1998—) ,女,硕士研究生,研究方向为配子发生的分子机制,E-mail: Luyueyee@ 126. com。
  • 基金资助:
    国家自然科学基金( 31970792)

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Abstract: Objective To construct mutant mouse with deletion of MIWI protein C-terminal and 3’UTR, and to investigate the effect of partial deletion on the function of MIWI protein. Method Mutant mice with partial deletion of MIWI protein was constructed by CRISPR / Cas9 gene editing technology. Firstly, fertility test was conducted to determine whether the mutation would lead to infertility in male mouse. Secondly, HE staining and other histological method were used to determine the time point of sperm development defects in the male mouse. Finally, the molecular defect of the mutant mice was investigated by Western blot and immunofluorescence staining. Result Fertility test showed that the Miwi + / Del2 heterozygous male mouse was completely infertile, sperm count showed that the total number of sperm decreased by 63%, Western blot showed that the MIWI protein decreased significantly. HE staining of the mutant testis sections showed that round spermatids were abnormal, with significantly increased abnormal sperm ( 74%) . Western blot and immunofluorescence staining results showed that protamine decreased significantly in testis, suggesting that protamine production and / or replacement were affected during sperm deformation, and normal long sperm could not be produced. Conclusion

A Miwi + / Del2 heterozygous mouse with partial deletion of Miwi C-terminal and 3’ UTR was successfully constructed. Deletion of C-terminal of MIWI protein can lead to male sterility.

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摘要：

目的 MIWI 蛋白C-端及3’ UTR部分缺失突变小鼠的构建,探究该部分缺失对MIWI蛋白功能的影响。方法 运用CRISPR / Cas9基因编辑技术构建了 MIWI 蛋白 C-端及 3’ UTR 部分缺失的雄鼠。首先,通过育性实验明确此突变是否会导致雄鼠不育。 其次,通过 HE 染色等组织学方法明确雄鼠精子发生缺陷出现的时间点。 最后,通过 Western blot 和免疫荧光染色探究该雄鼠不育的分子缺陷和潜在的分子生物学机制。 结果 育性实验结果显示 Miwi + / Del2 杂合雄鼠完全不育,精子计数显示其精子总数下降了 63%,Western blot 实验结果显示 Miwi + / Del2 杂合雄鼠中 MIWI 蛋白显著下降,HE 结果显示其圆形精子形态异常,精子涂片结果显示其畸形精子率为 74%,Western blot和免疫荧光染色结果显示睾丸鱼精蛋白下降显著,提示精子变形过程中鱼精蛋白产生和(或) 替换受影响,无法产生正常的长形精子。 结论 成功构建了一种 MIWI 蛋白 C-端及 3’ UTR 部分缺失的小鼠 Miwi + / Del2 。 MIWI 蛋白的C-端缺失可导致雄性不育。

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