Fancm基因敲除小鼠构建及其表型分析  

Generation and phenotypic analysis of Fancm gene knockout mice

作　　者：杨桥 郭红刚[1] 楼琦[1] 柯贤福[1] 周文伟[1] 应华忠[1] 俞冰[2] 张婷婷 YANG Qiao;GUO Honggang;LOU Qi;KE Xianfu;ZHOU Wenwei;YING Huazhong;YU Bing;ZHANG Tingting(Zhejiang Academy of Medical Sciences,Hangzhou 310000,China;Zhejiang School of Chinese Medicine,Hangzhou 310000)

机构地区：[1]浙江省医学科学院,杭州310000 [2]浙江中医药大学,杭州310000

出　　处：《中国实验动物学报》2019年第3期323-330,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：浙江省科技厅、卫生厅项目(2017F10007,2017C37142,2017C37157,2017KY037,2017KY038,2018C37125,2018KY342)~~

摘　　要：目的构建范可尼贫血通路Fancm基因敲除小鼠,研究Fancm基因缺失对小鼠生理功能,特别是雄性生殖器官的影响。方法采用CRISPR/Cas9技术,获得Fancm基因敲除小鼠。分析FANCM蛋白在野生型和Fancm^-/-小鼠睾丸组织中的表达。统计Fancm^-/-小鼠的出生率、体重、性别比例及子代生育情况,分析血液常规指标。组织形态学研究雄性Fancm^-/-小鼠睾丸生理病理表型。结果敲除Fancm基因ATG区域,获得稳定遗传的C57BL/6背景Fancm^-/-小鼠。Fancm^-/-小鼠睾丸中FANCM蛋白表达完全丢失。Fancm^-/-小鼠无明显的胚胎致死现象,但雌性Fancm-/-小鼠数目显著少于雄性Fancm^-/-小鼠。同窝Fancm^-/-小鼠比较野生型体重无明显区别,部分血常规指标有显著性差异。Fancm^-/-小鼠有明显的生殖能力缺陷。雄性Fancm^-/-小鼠睾丸有显著的发育缺陷,其生精细胞凋亡增加、细胞周期阻滞,影响睾丸发育与精子的生成。结论成功获得稳定遗传C57BL/6背景Fancm^-/-小鼠,Fancm基因参与小鼠的生长发育,特别是雄性生殖器官功能的维持及调控。Objective To generate a Fancm gene knockout mouse model and to elucidate the biological function of Fancm in mouse development.Methods Fancm^-/- mice were generated by the CRISPR/Cas9 method.We analyzed their body weight,birth rate,gender ratio,and fertility.Blood samples were analyzed by a complete blood count (CBC).Testes from the Fancm^-/- and wildtype littermates were subjected to pathophysiological assessment.Results C57BL/6 background Fancm knockout mice were generated by ATG region deletion using the CRISPR/Cas9 method.Western blotting indicated the complete depletion of FANCM protein in the testis lysate from Fancm^-/- mice.The Fancm^-/- mice showed no sign of embryonic lethality.However,there were significantly less female Fancm^-/- mice compared with male Fancm^-/- mice.There was no significant difference in body weight between the Fancm^-/- mice and wild type littermates.The CBC indicated a slight,but significant,increase in the Hb level.The Fancm^-/- mice were infertile.The male Fancm^-/- mice had a reduced testicular size and abnormal testicular architecture.The IHC and TUNEL assays indicated increases in cell cycle arrest and apoptosis in spermatogenic cells.Conclusions Fancm gene knockout causes defects of male reproductive organ development in mice.

关 键 词：范可尼贫血综合征 FANCM蛋白 Crispr/Cas9技术 Fancm基因敲除 睾丸发育缺陷 小鼠 

分 类 号：Q95-33[生物学—动物学]