Modified GFAP promoter auto-regulates tet-activator expression for increased transactivation and reduced tTA-associated toxicity
Modified GFAP promoter auto-regulates tet-activator expression for increased transactivation and reduced tTA-associated toxicity
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DOI：
10.1016/s0169-328x(02)00170-5
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发表时间：
2002-05-30
期刊：
影响因子：
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通讯作者：
Kwak, SP
中科院分区：
文献类型：
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作者：
Barton, MD;Dunlop, JW;Kwak, SP
反式激活因子 tTA 是四环素调节的诱导基因系统的必要组成部分。虽然已经描述了几种在中枢神经系统 (CNS) 中表达 tTA 的转基因动物，但它们的 tTA 水平通常受到限制，可能是由于毒性作用。我们评估了通过修改转基因启动子人 GFAP (hGFAP) 来自动调节星形胶质细胞中 tTA 水平的方法。携带单个tet操纵子拷贝的hGFAP启动子取代天然增强子元件（GFAPtetO1），在未刺激的基础条件下驱动tTA低水平表达。然而，在四环素停用后，相同的启动子自动诱导 tTA 表达至显着水平。在基础条件和诱导条件下，启动子的神经胶质细胞特异性均保持不变。使用自动诱导型 GFAPtetO1 启动子培育转基因大鼠，该启动子在大脑中高水平表达 tTA mRNA。表达在中枢神经系统内广泛存在，但在星形胶质细胞丰富的区域（包括小脑）富集。与 GFAP-tTA 转基因大鼠相比，转染 07LacZ 的 GFAPtetO1 大鼠的原代小脑星形胶质细胞产生显着更高的报告基因诱导能力。最后，当纯合性繁殖时，GFAPtetO1 大鼠表现出严重的运动/步态缺陷。这种表型可归因于小脑的发育异常，并且通过施用多西环素完全消除。这些结果表明，可以避免 tTA 表达引起的发育毒性，并且可以使用自激活策略开发具有高反式激活潜力的 tTA 转基因。这里提出的启动子修饰可能有助于开发高度诱导的转基因策略而不损失组织特异性。 (C) 2002 Elsevier Science B.V. 保留所有权利。
Transactivator tTA is a necessary component of the tetracycline-regulated inducible gene system. While several transgenic animals have been described that express tTA in the central nervous system (CNS), their tTA levels are often limited, presumably due to toxic effects. We evaluated methods for auto-regulating tTA levels in astrocytes by modifying the transgenic promoter human GFAP (hGFAP). The hGFAP promoter carrying a single copy of the tet-operon in place of a native enhancer element (GFAPtetO1) drove expression of tTA at low levels during un-stimulated, basal condition. However the same promoter auto-induced expression of tTA to significant levels after tetracycline withdrawal. Glial cell-specificity of the promoter remained uncompromised during both basal and induced conditions. Transgenic rats were developed using the auto-inducible GFAPtetO1 promoter that expressed tTA mRNA to high levels in the brain. Expression was widespread within the CNS but enriched in astrocyte-rich regions including the cerebellum. Primary cerebellar astrocytes from GFAPtetO1 rats transfected with 07LacZ produced substantially greater inducibility of reporter gene compared to GFAP-tTA transgenic rats. Finally, GFAPtetO1 rats exhibited severe motor/gait deficit when bred to homozygosity. This phenotype was attributable to developmental abnormalities of the cerebellum and was completely abrogated by doxycycline administration. These results suggest that developmental toxicity resulting from tTA expression can be circumvented and tTA transgenics with high transactivation potential can be developed using the auto-activation strategy. Promoter modification presented here may be useful in developing highly inducible transgenic strategies without loss in tissue-specificity. (C) 2002 Elsevier Science B.V. All rights reserved.