Adjusting transgene expression levels in lymphocytes with a set of inducible promoters
Adjusting transgene expression levels in lymphocytes with a set of inducible promoters
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DOI：
10.1002/jgm.1461
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发表时间：
2010-06-01
影响因子：
3.5
通讯作者：
Berens, Christian
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文献类型：
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作者：
Danke, Christina;Gruenz, Xandra;Berens, Christian
背景诱导基因表达系统是一种强大的研究工具，在未来可能具有临床价值，淋巴细胞可能是主要的应用靶点。然而，目前可用的可调控启动子以细胞系依赖性方式表现出其效率的变化，并且因基础渗漏或诱导性差而臭名昭著。关于淋巴细胞不同诱导型启动子的调控特性的数据很少。在本研究中，我们报告了各种诱导型启动子如何执行以及它们如何与transsilencer和反向反式激活因子组合可以导致T细胞中最佳控制的基因表达的综合分析。方法四环素调节（泰特）诱导型启动子Tet-responsive element（TRE），小鼠乳腺肿瘤病毒（MMTV）/TRE，为了监测含有转沉默子和反向反式激活子的Jurkat细胞系中的转基因表达，使用编码增强的绿色荧光蛋白的表达盒，CD 123或组成型活性的细胞毒性半胱天冬酶-3两侧有绝缘体并稳定整合。进一步分析了TREtight和SG/TRE在瞬时转染的原代CD 4（+）人T细胞中的性能。MMTV/TRE允许中度表达，TRE和TREtight允许中度表达，SG/TRE允许非常高的表达水平。TRE和SG/TRE是泄漏的，而MMTV/TRE和TREtight提供严格的表达控制。四环素衍生物通过引入中间表达水平增加了转基因表达的灵活性。结论启动子的不同表达谱增加了调节转基因表达水平的灵活性。启动子为许多应用提供了优化系统性能的附加选项。版权所有（C）2010约翰威利父子有限公司
Background Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells.Methods The performance of the tetracycline-regulated (Tet)-inducible promoters Tet-responsive element (TRE), mouse mammary tumor virus (MMTV)/TRE, TREtight and second generation TRE (SG/TRE) was compared in several B-cell lines and in Jurkat T-cells using transient transfections in combination with Tet-On. To monitor transgene expression in a Jurkat cell line containing a transsilencer and a reverse transactivator, expression cassettes encoding enhanced green fluorescent protein, CD123 or a constitutively active, cytotoxic caspase-3 were flanked with insulators and stably integrated. The performance of TREtight and SG/TRE was furthermore analysed in transiently transfected primary CD4(+) human T-cells.Results The promoters exhibit greatly diverging characteristics. MMTV/TRE permits moderate, TRE and TREtight permits intermediate and SG/TRE permits very high expression levels. TRE and SG/TRE are leaky, whereas MMTV/TRE and TREtight provide stringent expression control. Tetracycline derivatives add flexibility to transgene expression by introducing intermediate expression levels.Conclusions The different expression profiles of the promoters increase the flexibility to adjust transgene expression levels. The promoters provide an additional option to optimize system performance for many applications. Copyright (C) 2010 John Wiley & Sons, Ltd.