Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae
Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae
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DOI：
10.1128/ec.4.9.1539-1549.2005
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发表时间：
2005-09-01
期刊：
影响因子：
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通讯作者：
Marciniak, RA
中科院分区：
文献类型：
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作者：
McNabb, DS;Reed, R;Marciniak, RA
一种新的报告系统已被开发用于定量基因表达的酵母酿酒酵母。该系统依赖于两个不同的报告基因，海肾和萤火虫荧光素酶，以评估调控基因的表达。将编码海肾荧光素酶的基因与组成型启动子（PGK 1或SPT 15）融合，并整合到CAN 1基因座的酵母基因组中，作为标准化试验的对照。将萤火虫荧光素酶基因与测试启动子融合，并整合到酵母基因组的ura 3或leu 2基因座。通过依次测量同一样品的萤火虫和海肾荧光素酶活性来进行双荧光素酶测定，结果表示为萤火虫与海肾荧光素酶活性的比率（Fluc/Rluc）。酵母双荧光素酶报告基因（DLR）被表征并显示是非常有效的，需要大约1分钟来完成每个测定，并且已被证明产生准确且可再现地反映启动子活性的数据。产生了一系列整合质粒，其含有萤火虫或海肾荧光素酶基因，前面是两个不同方向的多克隆区和三个阅读框，以使翻译融合体的产生成为可能。此外，每组质粒含有用于酵母中遗传选择的URA 3或LEU 2标记。开发了一系列基于S288 C的酵母菌株，包括双杂交菌株，以促进酵母DLR测定的使用。该测定法可以很容易地适应于高通量平台，用于需要大量测量的研究。
A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately I minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.