Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system
Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system
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DOI：
10.1093/nar/gni030
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Daly, JM
中科院分区：
文献类型：
--
作者：
Voon, DC;Subrata, LS;Daly, JM
报告分析广泛应用于需要测量基因表达随时间变化的应用(例如药物筛选)。使用标准的报告载体，处理或化合物的可测量效果(改变的报告活性)与其真实效果(改变的转录活性)相比被大大稀释和延迟。这个问题是由报告蛋白及其mRNA的相对较长的半衰期引起的。因此，具有相对较小影响或重大但短暂影响的化合物、配体或处理方法的活性通常仍未被检测到。为了绕过这个问题，我们在一系列常用的记者中引入了模块化的蛋白质和mRNA破坏稳定的元件。我们的数据显示，这两个元件都是对转录活性增加和减少做出最大反应所必需的。双失稳报告载体在药物筛选、动力学分析和剂量-反应滴定等方面表现出明显的改善。
Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose-response titrations.