基于Cre/loxP系统的睾丸组织特异性敲除Elovl4基因小鼠模型的构建及敲除效率检测  

Construction of a testis Elovl4 gene knockout mouse model based on Cre/loxP system

作　　者：杨仕赛 赵瑄 王雨虹 郑红梅 甘婷 朱贵明 YANG Shisai;ZHAO Xuan;WANG Yuhong;ZHENG Hongmei;GAN Ting;ZHU Guiming(School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Biological and Medical Engineering,School of Biology and Engineering,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院,贵州贵阳550025 [2]贵州医科大学生物与工程学院生物与医学工程重点实验室,贵州贵阳550025

出　　处：《生物工程学报》2022年第8期2912-2927,共16页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(31960205);贵州省教育厅创新群体重大研究项目(黔教合KY字[2016]031)。

摘　　要：极长链多不饱和脂肪酸(very long chain polyunsaturated fatty acids,VLC-PUFAs)是哺乳动物视网膜、睾丸等极少数组织中特有的脂肪酸,其生物合成的关键酶为极长链脂肪酸延长酶4(very long chain fatty acid elongase 4,Elovl4)。建立组织特异性敲除Elovl4基因的动物模型有利于深入研究VLC-PUFAs的生物学功能,因此,本研究基于Cre/loxP系统,先分别构建了Stra8-Cre小鼠和Elovl4 floxed小鼠,通过杂交获得(Elovl4[flox/+],Stra8-Cre)杂合子基因敲除小鼠,再选择雌鼠与Elovl4 floxed纯合子雄鼠即Elovl4[flox/flox]雄鼠杂交,通过基因型鉴定筛选获得(Elovl4[flox/flox],Stra8-Cre)纯合子小鼠。利用RT-PCR、qRT-PCR、Western blotting、免疫组化和免疫荧光检测Elovl4在睾丸组织中的敲除效率,结果表明,无论是杂合子还是纯合子基因敲除小鼠,其睾丸组织中Elovl4的表达在mRNA及蛋白水平显著下调,但其他组织未受影响。本研究成功构建了睾丸组织特异性敲除Elovl4基因小鼠,为后续研究VLC-PUFAs对雄性小鼠生殖功能的影响及相关分子机制提供可靠的动物模型。Very long chain polyunsaturated fatty acids(VLC-PUFAs)are unique fatty acids in tissues of mammals such as retina and testis,and the key enzyme of its biosynthesis is very long chain fatty acid elongase 4(Elovl4).Development of an animal model of tissue-specific knockout of Elovl4 gene is conducive to the in-depth study of the biological function of VLC-PUFAs.Therefore,we constructed Stra8-Cre mice and Elovl4 floxed mice based on Cre/loxP system,and obtained the(Elovl4[flox/+],Stra8-Cre)heterozygous knockout mice by hybridization.Subsequently,female mice were selected to cross with male mice with homozygous Elovl4[flox/flox]to gain homozygous mice(Elovl4[flox/flox],Stra8-Cre)through genotype identification and screening.RT-PCR,qRT-PCR,Western blotting,immunohistochemistry and immunofluorescence techniques were used to detect the knock-out efficiency of Elovl4 in testis.The expression of Elovl4 in testis of both heterozygous and homozygous knockout mice were significantly down-regulated at mRNA and protein levels,but were not affected in other tissues.In summary,we constructed a mouse model with specific knockout of Elovl4 gene in testis,which provides a reliable animal model for studying the effect of VLC-PUFAs on the reproductive function of male mice and the underpinning molecular mechanisms.

关 键 词：Cre/loxP 睾丸 Elovl4 Stra8-Cre 特异性敲除 

分 类 号：Q78[生物学—分子生物学]