精子特异性锌指蛋白146条件性敲除小鼠的构建及鉴定

Construction and Authentication of the Sperm-Specific RNF146 Conditional Knockout Mice

目的探讨构建精子特异性RNF146敲除小鼠及其鉴定方法。方法从NCBI网站上检索小鼠RNF146的基因序列,分析外显子与各剪接体的关系以及起始密码子和终止密码子所在区域,选择条件性基因敲除片段。构建基于Cre/Lox P系统的RNF146条件性敲除杂合子(RNF146^(flox/+))小鼠(F1代)。取F1代RNF146^(flox/)+杂合子小鼠自交,PCR鉴定其后代基因型,挑选RNF146纯合子敲除(RNF146^(flox/flox))雌鼠与精子特异性Cre转基因雄鼠AQP2-Cre进行杂交。PCR检测其后代基因型,选取AQP2-Cre阳性的RNF146杂合子雄鼠(AQP2-Cre;RNF146^(flox/+))与RNF146纯合子(RNF146^(flox/flox))雌鼠回交后筛选AQP2-Cre阳性的RNF146纯合子小鼠(AQP2-Cre;RNF146^(flox/flox))即为精子特异性RNF146敲除小鼠。选取9~10周龄(体成熟)的精子特异性RNF146敲除小鼠,从附睾和输精管中分离精子,以AQP2-Cre;RNF146^(flox/+)小鼠为对照,Real-time PCR及Western blot分析精子中RNF146表达水平确认敲减效率。结果RNF146基因四号外显子被选为敲除区域来构建基于Cre/Lox P系统的条件性敲除小鼠。F1代杂合子(RNF146^(flox/+))小鼠自交后代中约1/4个体为RNF146^(flox/flox);AQP2-Cre转基因工具鼠与纯合子(RNF146^(flox/flox))雌鼠交配后代中具有AQP2-Cre;RNF146^(flox/+)基因型的小鼠约占1/2;AQP2-Cre;RNF146^(flox/+)雄鼠和RNF146^(flox/flox)雌鼠交配后代中近1/4为AQP2-Cre;RNF146^(flox/flox)小鼠。成年AQP2-Cre;RNF146^(flox/flox)小鼠精子中RNF146 m RNA(Real-time PCR)和蛋白表达水平(Western blot)分别降低77.3%(P<0.001)和89.2%(P<0.001)。结论成功构建精子特异性RNF146条件性敲除小鼠AQP2-Cre;RNF146^(flox/flox)。

Objective To construct the sperm-specific zinc finger protein 146(RNF146)conditional knockout mice and confirm its efficiency of knockdown in mouse sperms.Methods The gene sequence of mouse RNF146 was acquired on the NCBI website.According to the relationship between its exons and transcript variants and the region of the exons including the start codon and the stop codon,the conditional knockout region of mouse RNF146 was determined.RNF146 conditional knockout heterozygous(RNF146flox/+)mice(offspring F1)based on the Cre/LoxP system were constructed by the Cyagen company.After F1 mice bred with each other,the genotypes of offspring F1 were authenticated using PCR.The RNF146flox/flox male bred with sperm-specific Cre transgenic females(AQP2-Cre)to reproduce the AQP2-Cre positive and RNF146flox/+male(AQP2-Cre;RNF146flox/+)in offspring F3,which further bred with RNF146 homozygous(RNF146flox/flox)females.In offspring F4,AQP2-Cre positive and RNF146flox/flox males(AQP2-Cre;RNF146flox/flox)were employed as the sperm-specific RNF146 conditional knockout mice in this study.Sperms were isolated from epididymis and vas deferens of the 9-10 weeks age sperm-specific RNF146 knockout mice and the RNF146 expression in the isolated sperms were analyzed using Real-time PCR and Western blot to confirm the knockout efficiency of RNF146 in the mouse sperms.Results The exon 4 of RNF146 gene was determined as the knockout region.A quarter of the offspring mice from RNF146flox/+mice was euthanticated as RNF146flox/flox.Nearly half of the offsprings from the AQP2-Cre transgenic mice bred with RNF146flox/flox were the AQP2-Cre;RNF146flox/+mice.Around a quarter of the offsprings from the AQP2-Cre;RNF146flox/+male bred with the RNF146flox/flox mice was authenticated as AQP2-Cre;RNF146flox/flox mice.RNF146 mRNA expression decreased by 77.3%(P<0.001)and protein levels decreased 89.2%(P<0.001)using Real-time PCR and Western blot detection,respectively.Conclusion A sperm-specific RNF146 conditional knockout mice AQP2-Cre;RNF146flox/flox was successfully constructed.