Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators.
Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators.
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DOI：
10.1186/1471-2121-10-14
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发表时间：
2009-02-25
期刊：
影响因子：
--
通讯作者：
Link W
中科院分区：
文献类型：
--
作者：
Zanella F;Rosado A;Garcia B;Carnero A;Link W
独立的荧光素酶报告基因测定和荧光易位测定已成功地用于几种分子靶点的药物发现。我们开发了U2 transLUC，这是一种检测系统，其中荧光素酶和荧光读数可以多重化，以提供强大的基于细胞的高含量筛选方法。U2 transLUC系统基于在人FOXO应答增强子控制下表达GFP标记的FOXO转录因子和荧光素酶报告基因的稳定细胞系。U2 transLUC测定法测量核质FOXO穿梭和FOXO驱动的转录，提供了在同一实验中分析FOXO调节的这两个关键特征的方法。我们用具有已知生物活性的化学探针挑战U2 transLUC系统，并且我们能够鉴定具有易位和/或反式激活能力的化合物。在单个细胞系中组合不同的生物学读数提供了优于常规的基于细胞的测定的显著优势。U2 transLUC检测有助于维持和监测同质FOXO转录因子表达，并允许测量的报告基因活性相对于细胞活力进行标准化。U2 transLUC适用于高通量筛选，可以识别不同水平干扰FOXO信号传导的小分子。
Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method. The U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers. The U2transLUC assay measures nuclear-cytoplasmic FOXO shuttling and FOXO-driven transcription, providing a means to analyze these two key features of FOXO regulation in the same experiment. We challenged the U2transLUC system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity. Combining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. The U2transLUC assay facilitates the maintenance and monitoring of homogeneous FOXO transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.
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影响因子：
4
作者：
Huang, Haojie;Tindall, Donald J.
通讯作者：
Tindall, Donald J.
影响因子：
3.7
作者：
Rosado, Aranzazu;Zanella, Fabian;Link, Wolfgang
通讯作者：
Link, Wolfgang
影响因子：
4.8
作者：
Tsai, WC;Bhattacharyya, N;Rechler, MM
通讯作者：
Rechler, MM
影响因子：
1.8
作者：
Zanella, Fabian;Rosado, Aranzazu;Link, Wolfgang
通讯作者：
Link, Wolfgang
DOI：
10.1073/pnas.0502738102
发表时间：
2005-08-09
影响因子：
11.1
作者：
Matsuzaki, H;Daitoku, H;Fukamizu, A
通讯作者：
Fukamizu, A