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李卫杰,梁冬丽,刘永忠,王朝霞,乔中东,徐汪节.生殖细胞条件性敲除 Usp16 基因小鼠的建立、鉴定及表型分析[J].中国实验动物学报,2022,30(3):376~383.
生殖细胞条件性敲除 Usp16 基因小鼠的建立、鉴定及表型分析
Establishment, identification and phenotyping of a germ cell-specific deubiquitinating enzyme 16 conditional knockout mouse
投稿时间:2021-09-24  
DOI:10. 3969 / j.issn.1005-4847. 2022. 03. 010
中文关键词:  精子发生  Usp16  生殖力  Cre-loxP
英文关键词:spermatogenesis  Usp16  fertility  Cre-loxP
基金项目:
作者单位E-mail
李卫杰 上海交通大学生命科学技术学院,上海 200240 lwj897586647@ sjtu. edu. cn 
梁冬丽 上海交通大学实验动物中心,上海 200240  
刘永忠 上海交通大学医学院仁济医院上海肿瘤研究所癌基因与相关基因国家重点实验室,上海 200240  
王朝霞 上海交通大学实验动物中心,上海 200240  
乔中东 上海交通大学生命科学技术学院,上海 200240  
徐汪节 上海交通大学实验动物中心,上海 200240 hover_xwj@ sjtu. edu. cn 
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中文摘要:
       目的 建立生殖细胞特异性敲除去泛素化酶 Usp16 小鼠,并分析其生殖表型,探究 Usp16 是否在精子发生过程发挥功能。 方法 首先,将 Vasa-cre 转基因小鼠与 Usp16 条件性小鼠(Usp16flox / flox )杂交获得生殖细胞特异性敲除 Usp16 小鼠;其次,利用 qPCR、Western Blot 和免疫荧光检测睾丸中 Usp16 表达;并通过合笼交配、计算机辅助精子分析和 HE 染色法进行生殖表型分析。 结果 成功获得了生殖细胞特异性敲除 Usp16 纯合子 (Usp16flox / flox / Vasa-cre+ ),及杂合子小鼠(Usp16flox / wt / Vasa-cre+ )。 与野生型相比,Usp16flox / wt / Vasa-cre+雄鼠生殖表型没有显著性变化,但与纯合子雄鼠交配的雌鼠受孕率仅为 7%,精子数量减少约 79%,活力下降约 87%,精子畸形率高达 83%,睾丸曲细精管中出现多核细胞,附睾中早熟精子数增多了约 3 倍。 结论 小鼠睾丸内生殖细胞 Usp16 纯合敲除会严重降低精子质量,从而降低雄性生育能力。
英文摘要:
       Objective To study the role of deubiquitinating enzyme 16 ( Usp16 ) in spermatogenesis by establishing a mouse model of conditional Usp16 knockout in germ cells. Methods Vasa-cre transgenic mice were crossed with Usp16 transgenic mice (Usp16flox / flox) to obtain germ cell-specific Usp16 knockout mice. To confirm that Usp16 had been conditionally inactivated in germ cells, genotyping by PCR and western blotting was performed to evaluate Usp16 expression. Initial characterization of the phenotype of conditional knockout (CKO) mice was then conducted by co-cage mating, computer-assisted sperm analysis, and HE staining. Results We confirmed that Usp16 expression was appropriate in the conditional knockout mouse. Compared with the wild type, the conception rate of female mice mated with CKO mice had decreased to only 7%, while no significant difference was observed in heterozygous knockout mice male mice. The sperm count of CKO mice was decreased by approximately 79%, motility was decreased by approximately 87%, and the rate of sperm deformity was as high as 83%. Multinucleated cells appeared in the seminiferous tubules of the testes and the number of precocious sperm in the epididymis had increased by approximately 76%. Conclusions Homozygous mutation of Usp16 in germ cells severely reduces sperm quality, thereby reducing male fertility.
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