【摘要】 现有的四环素诱导调控系统基于两个单独的质粒分别表达反式结合蛋白和外源基因.其缺点是在建立转基因定量表达动物模型时,需要制备和维持两个动物品系,再进行杂交才有可能获得双转基因后代,步骤繁琐,难度较大.针对上述缺陷,本研究尝试将反式蛋白rtTA表达框和低背景响应元件Ptight组装到同一个载体上,构建为严谨型单载体模式的诱导表达系统pTRE-Tight-rtTA,并通过两种报告基因的表达对其调控活性进行了研究.含有荧光素酶和绿色荧光蛋白的pTRE-Tight-rtTA-Luc和pTRE-Tight-rtTA-EGFP报告载体分别转染猪肾PK15细胞并经强力霉素处理,均可成功诱导报告基因的定量表达.在等摩尔转染条件下,单载体系统的诱导效率明显高于双载体系统(Dox-1 000 ng,10倍;Dox-10 000 ng,8倍).该诱导型单载体系统的成功构建为外源基因的定量表达提供了新手段,为转基因定量表达动物模型的研究提供了新策略.更多还原

【Abstract】 The tetracycline-inducible gene regulation system includes two individual plasmids to express rtTA binding protein and gene of interest.A disadvantage of this dual-plasmid system is that when it is applied to transgenic quantitative expression animal model,two lines of animals have to be constructed and maintained.Hybridization is required to produce genetically modified animals harboring both rtTA and gene of interest.The procedures are complicated,time-consuming and labor-intensive.To solve this problem,we constructed tetracycline-inducible single-plasmid system that rtTA binding protein and low-background responsive element Ptight promoter are assembled into one vector.Its regulatory activity is analyzed by measuring the expression of reporter gene.EGFP and luciferase expression can be quantitatively induced when they were transfected into pig kidney PK15 cell lines and induced by doxycycline.In identical molar ratio,the induction efficiency of single-plasmid is much higher than dual-plasmid.This inducible single-plasmid adds a new tool for foreign gene quantitative expression,and it also is a novel strategy to construct transgenic quantitative expression animal model.更多还原