一种基于HAC1的新型双荧光素酶报告载体，用于检测酿酒酵母中的内质网应激和未折叠蛋白反应。
A novel HAC1-based dual-luciferase reporter vector for detecting endoplasmic reticulum stress and unfolded protein response in yeast Saccharomyces cerevisiae.
作者信息
Fang Zhijia, Kuang Xin, Zhang Youshang, Shi Ping, Huang Zhiwei
机构信息
Key Lab of Eco-Textile, Ministry of Education, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 Renmin Road, Shanghai 201620, China.
Key Lab of Eco-Textile, Ministry of Education, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 Renmin Road, Shanghai 201620, China; Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China.
出版信息
Plasmid. 2015 May;79:48-53. doi: 10.1016/j.plasmid.2015.04.002. Epub 2015 Apr 20.
Unfolded protein response (UPR) is an important cellular phenomenon induced by over-accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen. ER stress and UPR are implicated in human diseases such as diabetes, atherosclerosis and neurodegenerative diseases. Current methods for measuring ER stress levels and UPR activation usually include cells lysis and other complicated procedures such as reverse transcription-PCR (RT-PCR). These methods typically have low sensitivity and are not suitable for live detection. In this study, we developed a dual-luciferase gene reporter system to monitor UPR activation in live cells of the yeast Saccharomyces cerevisiae by taking advantage of the HAC1 intron and its unconventional splicing-regulation mechanism. We showed that this reporter can be used to monitor UPR in live cells with high sensitivity.
未折叠蛋白反应（UPR）是内质网（ER）腔中未折叠蛋白过度积累所诱导的一种重要细胞现象。内质网应激和未折叠蛋白反应与糖尿病、动脉粥样硬化和神经退行性疾病等人类疾病有关。目前测量内质网应激水平和未折叠蛋白反应激活的方法通常包括细胞裂解以及其他复杂程序，如逆转录聚合酶链反应（RT-PCR）。这些方法通常灵敏度较低，不适用于实时检测。在本研究中，我们利用HAC1内含子及其非常规剪接调控机制，开发了一种双荧光素酶基因报告系统，用于监测酿酒酵母活细胞中的未折叠蛋白反应激活。我们表明，该报告系统可用于高灵敏度地监测活细胞中的未折叠蛋白反应。
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