利用PiggyBac转座子系统构建表达Δ15 Des酶活性的转基因小鼠  

Construction of transgenic mice with Δ15 Des enzyme activity by using a PiggyBac transposon

作　　者：王颖 杨仕赛 赵瑄 李亚 吕露露 朱贵明 WANG Ying;YANG Shisai;ZHAO Xuan;LI Ya;LÜ Lulu;ZHU Guiming(Key Laboratory of Biology and Medical Engineering,College of Biology and Engineering,Guizhou Medical University,Guiyang 550025,Guizhou,China;Engineering Research Center of Medical Biotechnology,College of Biology and Engineering,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]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 [2]贵州医科大学生物与工程学院医药生物技术工程研究中心,贵州贵阳550025

出　　处：《生物工程学报》2022年第1期196-206,共11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：必需脂肪酸为人体健康和生命所必需,但机体自身不能合成,研究表明ω-3族脂肪酸对人体的生理功能有更加积极的影响。哺乳动物体内缺乏ω-3去饱和酶的基因,来自线虫Caenorhabditis elegans的Δ15-脂肪酸去饱和酶(Δ15Des)可以将体内ω-6多不饱和脂肪酸(polyunsaturated fatty acids, PUFAs)转化为ω-3 PUFAs。利用PiggyBac转座子(PB)系统构建表达Δ15 Des酶活性的转基因小鼠可以在较短时间内繁育出稳定遗传的纯合子转基因小鼠,整合率高达35.1%。饲料中添加6%ω-6 PUFAs饲喂小鼠,通过气相色谱(gas chromatography, GC)检测小鼠体内脂肪酸的变化,荧光定量PCR(quantitative PCR,qPCR)和蛋白免疫印迹(Western blotting, WB)来检测Δ15 Des在小鼠体内的表达水平。从qPCR和GC结果分析,阳性小鼠的基因活性率为61.53%,与传统方法相比,Δ15 Des的转入效率及活性都显著提高,且纯合子比杂合子表达更高的活性,进一步验证了PiggyBac转座子系统高效的转导效率和安全稳定性。Essential fatty acids are those that could not be synthesized by the body itself but crucial for health and life. Studies have shown that ω-3 fatty acids may facilitate human physiological functions.Mammals lack ω-3 desaturase gene, and the Δ15 fatty acid desaturase(Δ15 Des) from Caenorhabditis elegans can transform the ω-6 polyunsaturated fatty acids(PUFAs) into ω-3 PUFAs. Transgenic mice expressing Δ15 Des enzyme activity was constructed by using a PiggyBac transposon(PB). Homozygous transgenic mice with stable inheritance was bred in a short time, with a positive rate of 35.1% achieved. The mice were fed with 6% ω-6 PUFAs and the changes of fatty acids in mice were detected by gas chromatography(GC). The expression level of Δ15 Des in mice was detected by quantitative PCR(qPCR)and Western blotting(WB). qPCR and GC analysis revealed that the percentage of positive mice harboring the active gene was 61.53%. Compared with traditional methods, the transformation efficiency and activity of Δ15 Des were significantly improved, and homozygotes showed higher activity than that of heterozygotes.This further verified the efficient transduction efficiency of the PiggyBac transposon system.

关 键 词：多不饱和脂肪酸 Δ15脂肪酸去饱和酶 转座子 转基因 

分 类 号：Q78[生物学—分子生物学]