Improved dual-luciferase reporter assays for nuclear receptors.
Improved dual-luciferase reporter assays for nuclear receptors.
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DOI：
10.2174/1875397301004010043
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Fan F
中科院分区：
文献类型：
--
作者：
Paguio A;Stecha P;Wood KV;Fan F
核受体通过控制基因转录在许多细胞功能中发挥重要作用。它也是药物发现的一个大目标类别。荧光素酶报告基因检测因其动态范围宽、内源活性低且易于使用而经常用于研究核受体功能。荧光素酶基因和载体的最新改进进一步增强了它们的效用。在这里，我们将这些改进应用于两种报告基因格式以研究核受体。第一个测定包含不稳定的荧光素酶上游的鼠乳腺肿瘤病毒启动子。该启动子中核激素受体反应元件的存在允许内源和/或外源全长受体的研究。第二个测定包含与一个载体上的 GAL4 DNA 结合域 (DBD) 融合的核受体的配体结合域 (LBD) 和另一个载体上荧光素酶报告基因上游的多个 Gal4 上游激活序列 (UAS)。我们表明，荧光素酶报告基因的密码子优化增加了表达水平，同时将蛋白质不稳定序列掺入荧光素酶中，导致两种形式的检测动态范围更大。确定了产生最佳响应的 UAS 的最佳数量。核受体LBD/GAL4 DBD融合的表达载体还组成型表达海肾荧光素酶-neoR融合蛋白，其提供选择能力（G418抗性，neoR）以及内部对照（海肾荧光素酶）。这种双荧光素酶格式允许在药物筛选过程中检测化合物的细胞毒性或表达的脱靶变化，从而提高数据质量。这些荧光素酶报告基因检测为研究全长核受体和配体结合域的功能提供了更好的研究和药物发现工具。
Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neoR fusion protein, which provides selection capability (G418 resistance, neoR) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains.