Spectral-Resolved Gene Technology for Multiplexed Bioluminescence and High-Content Screening

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• 作者：Elisa Michelini ; Luca Cevenini ; Laura Mezzanotte ; Danielle Ablamsky ; Tara Southworth ; Bruce Branchini ; Aldo Roda
• 刊名：Analytical Chemistry
• 出版年：2008
• 出版时间：January 1, 2008
• 年：2008
• 卷：80
• 期：1
• 页码：260 - 267
• 全文大小：132K
• 年卷期：v.80,no.1(January 1, 2008)
• ISSN：1520-6882

文摘

The availability of new bioluminescent proteins, obtainedby cDNA cloning and mutagenesis of wild-type genes,expanded the applicability of these reporters from theperspective of using more proteins emitting at differentwavelengths in the same cell-based assay. By spectrallyresolving the light emitted by different reporter proteins,it is in fact possible to simultaneously monitor multipletargets. A new luciferase isolated from Luciola italicahas been recently cloned, and thermostable red- andgreen-emitting mutants were obtained by random and site-directed mutagenesis. Different combinations of luciferases were used in vitro as purified proteins and expressedin bacterial and mammalian cells to test their suitabilityfor multicolor assays. A mammalian triple-color reportermodel system was then developed using a green-emittingwild-type Photinus pyralis luciferase, a red thermostablemutant of L. italica luciferase, and a secreted Gaussiaprinceps luciferase (GLuc) to monitor the two main pathways of bile acid biosynthesis. The two firefly luciferaseswere used to monitor cholesterol 7- hydroxylase andsterol 27-hydroxylase, while secreted constitutively expressed GLuc was used as an internal vitality control. Bytreating the cells with chenodeoxycholic acid, it waspossible to obtain dose-dependent inhibitions of the twospecific signals together with a constant production ofGLuc, which allowed for a dynamic evaluation of themetabolic activity of the cells. This is the first triple-colormammalian reporter assay that combines secreted andnonsecreted luciferases requiring different substrates,thus avoiding reciprocal interference between different BLsignals. This approach is suitable for high content analysisof gene transcription in living cells to shorten the time forscreening assays, increasing throughput and cost-effectiveness.