Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis.
Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis.
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DOI：
10.1158/0008-5472.can-14-3538
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发表时间：
2015-12-01
期刊：
影响因子：
11.2
通讯作者：
Amelio AL
中科院分区：
文献类型：
--
作者：
Schaub FX;Reza MS;Flaveny CA;Li W;Musicant AM;Hoxha S;Guo M;Cleveland JL;Amelio AL
荧光蛋白被广泛用于研究分子和细胞事件，但这传统上依赖于激发光的传递，而激发光会引发自身荧光、光毒性和光漂白，从而损害其在体内的使用。因此，由荧光素酶产生的化学发光光源出现了，因为它们不需要激发光。然而，目前的荧光素酶报告缺乏可视化深层组织事件所需的亮度。我们报道了嵌合eGFP-NanoLuc （GpNLuc）和LSSmOrange-NanoLuc （OgNLuc）融合报告蛋白LumiFluors的创建，该蛋白将eGFP或LSSmOrange荧光蛋白的优点与深海虾（Oplophorus gracilirostris）增强的小荧光素酶亚基（NanoLuc）产生的明亮，发光型生物发光结合起来。发生在NanoLuc和融合荧光团之间的分子内生物发光共振能量转移（BRET）产生迄今为止已知的最亮的生物发光信号，包括改进的强度、灵敏度和持久的光谱特性，从而大大减少了图像采集时间，并允许高灵敏度的体内成像。值得注意的是，这些LumiFluor报告器的自发光和双功能特性可以通过体内光学成像大大改善对极少数肿瘤细胞的时空监测，还可以通过流式细胞术分离和分析单个细胞。因此，LumiFluor报告器是一种廉价、强大、非侵入性的工具，可以显著改善肿瘤发生过程的体内光学成像。
Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer (BRET) that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bi-functional nature of these LumiFluor reporters enables greatly improved spatio-temporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, non-invasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.