Michelini Elisa, Cevenini Luca, Mezzanotte Laura, Ablamsky Danielle, Southworth Tara, Branchini Bruce R, Roda Aldo
Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126, Bologna, Italy.
Photochem Photobiol Sci. 2008 Feb;7(2):212-7. doi: 10.1039/b714251j. Epub 2008 Jan 14.
Bioluminescent (BL) proteins are a promising tool for diverse applications based on reporter gene technology thanks to their high sensitivity and range of linear response. Due to their widespread use in the environmental, medical and agro-food fields, there is a great need for new BL reporter proteins with improved characteristics to provide researches a wide range of suitable reporters. Few efforts have been made in this direction and further improvement of BL reporter features (e.g., thermostability, narrower emission bandwidth, emission at different wavelengths) tailored for specific applications would be a remarkable progress toward the development of ultrasensitive multiplexed assays either in vitro or in vivo. The suitability of using red- and green-emitting thermostable mutants of Photinus pyralis firefly luciferase and two click beetle luciferases in combination with a secreted luciferase from Gaussia princeps was evaluated to develop a triple-color mammalian assay. Two triple-reporter model mammalian systems were developed in a human hepatoblastoma cell line to monitor the transcriptional regulation of cholesterol 7-alpha hydroxylase (cyp7a1), the enzyme that catalyzes the first and rate-limiting step of the main pathway responsible for cholesterol degradation in humans. These model systems allowed us to evaluate the feasibility of using two intracellular BL reporters and a secreted one in the same cell-based assay. The selection of reporter proteins characterized by similar expression levels was identified as a critical point for the development of a multicolor assay.
生物发光（BL）蛋白因其高灵敏度和线性响应范围，是基于报告基因技术的多种应用的有前途的工具。由于它们在环境、医学和农业食品领域的广泛应用，迫切需要具有改进特性的新型BL报告蛋白，为研究人员提供广泛合适的报告蛋白。在这个方向上所做的努力很少，针对特定应用定制的BL报告特性（例如，热稳定性、更窄的发射带宽、不同波长的发射）的进一步改进将是朝着开发体外或体内超灵敏多重检测方法迈出的显著进展。评估了使用萤火虫荧光素酶和两种叩甲荧光素酶的红色和绿色发光热稳定突变体与来自王子高斯藻的分泌型荧光素酶相结合来开发三色哺乳动物检测方法的适用性。在人肝癌细胞系中开发了两个三报告基因模型哺乳动物系统，以监测胆固醇7-α羟化酶（cyp7a1）的转录调控，该酶催化人类胆固醇降解主要途径的第一步和限速步骤。这些模型系统使我们能够评估在同一基于细胞的检测中使用两种细胞内BL报告蛋白和一种分泌型报告蛋白的可行性。选择具有相似表达水平的报告蛋白被确定为开发多色检测方法的关键点。
