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

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。

Abstract:

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.