时空调控 miR-195敲减转基因载体的构建及验证  

Construction and identification of miR-1 9 5 silencing transgenic vector with temporal and spatial regulation

作　　者：班涛[1] 张玉瑶[2] 吴兆菊[1] 赵欣[3] 朱久新[1] 

机构地区：[1]哈尔滨医科大学药学院药理教研室,150081 [2]黑龙江中医药大学基础医学院解剖与组胚教研室,150040 [3]哈尔滨市第一医院药剂科,150010

出　　处：《国际遗传学杂志》2016年第3期123-132,共10页International Journal of Genetics

基　　金：黑龙江省教育厅科学技术研究项目面上项目（12531252）

摘　　要：目的：构建时空调控 miR-195敲减转基因载体并验证其活性。方法通过分子克隆技术构建含有神经特异性启动子、四环素诱导表达系统、miR-195海绵、绿色荧光蛋白及绝缘子等调控元件的转基因载体并测序鉴定，利用细胞转染技术将该载体瞬时转染入 SH-SY 5 Y 细胞，经荧光显微镜观察其荧光表达，利用实时定量 PCR 技术及蛋白免疫印迹检测miR-195含量及 GFP 蛋白表达。结果通过将 Tet-on 四环素诱导系统中反义转录激活子 rt-TA 克隆到 NSE 启动子下游，以及 TRE-Tight 的多克隆位点内引入 miR-195海绵携带 IRES 介导的 EGFP ，获得双质粒 Tet-on 表达载体。以 pcDNA 3.1＋质粒为骨架，3个不同区域的双拷贝 cHs 4绝缘子核心片段为间隔，将其调控元件（ NSE-rtTA ）和反应元件（ TRE-miR-195 sponge-IRES-EGFP ）串联成单一转基因载体。该载体经测序验证序列正确，瞬时转染入 SH-SY 5 Y 细胞，经强力霉素诱导36 h 后细胞内可见明显绿色荧光蛋白表达，且 miR-195表达水平明显降低。结论成功构建神经系统特异性 miR-195敲减转基因载体，并具有时空调控的特点，为进一步建立转基因动物模型奠定基础。Objective The purpose of this study is to construct a validated miR-1 9 5 silencing transgenic vector with temporal and spatial regulation . Methods Five different regulatory elements （ a neuron-specific promoter , Tet-on tetracycline inducible expression system , miR-1 9 5 sponge , green fluo-rescent protein and cHs 4 insulators ） separately were used to construct the transgenic vector by molecular cloning techniques .After a sequencing confirmation , the newly established vector was transiently transfect-ed in SH-SY 5 Y cells; the transfection efficiency was confirmed by fluorescence microscopy ; Western blot showed the GFP protein expression . MiR-1 9 5 expression level was measured by real-time quantitative PCR .Results The reverse Tet Transactivator （ rtTA ） element of the Tet-on advanced system was cloned into the downstream of the NSE promoter , and the fragment of the miR-1 9 5 sponge that carrying IRES-mediated EGFP was constructured into the multiple cloning site of the TRE -Tight vector .The regulatory＆amp;nbsp;element （ NSE-rtTA ） and the response element （ TRE-miR-1 9 5 sponge-IRES-EGFP ） were introduced in a pcDNA 3 .1 ＋backbone with intervals by three different regional 2 × cHs 4 insulator core fragments to construct a neuron-specific miR-1 9 5 silencing transgenic vector .The vector was transiently cotransfected with miR-1 9 5 in the SH-SY 5 Y cells treated with Doxycycline inducers .The green fluorescence was visi-ble in the cytoplasm of the treated cells and the GFP proteins can be detected , and the miR-1 9 5 level was also significantly reduced compare with the miR-1 9 5 transfected cells .Conclusion Neuron-specific miR-1 9 5 silencing transgenic vector was successfully constructed .The vector exhibited concurrent temporal and spatial regulation characteristic in vitro .The successful construction will lay foundations for the estab-lishment of transgenic mouse models .

关 键 词：miR-195 转基因载体 Tet-on四环素诱导表达系统 

分 类 号：Q78[生物学—分子生物学]