A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.
A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.
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DOI：
10.1007/s00216-014-7917-2
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发表时间：
2014-09
影响因子：
4.3
通讯作者：
Kaijzel, Eric L.
中科院分区：
文献类型：
--
作者：
Mezzanotte, Laura;Blankevoort, Vicky;Lowik, Clemens W. G. M.;Kaijzel, Eric L.
根据应用的不同，荧光成像和生物发光成像各有优缺点。生物发光成像现在是跟踪细胞、启动子活性研究或体内纵向临床前研究的最灵敏的光学技术。由于有非常灵敏的成像仪器，远红外和近红外荧光成像具有既适用于体外分析又适用于体内分析的优点，并具有转换潜力。在这里，我们报道了一种新的荧光素酶融合报告的开发和验证，该融合报告是通过一个14个氨基酸的连接肽将萤火虫荧光素酶Luc2与远红荧光蛋白TurboFP635融合而产生的。通过Western印迹分析、荧光显微镜和体内光学成像，分析了融合蛋白TurboLuc在人胚胎肾脏细胞(HEK)-293细胞中的表达。产生的融合蛋白保持了原始生物发光和荧光蛋白的特性，在活细胞中表达时没有毒性。为了评估该报告对活体成像的敏感性，将转基因细胞注射到动物的皮下。生物发光和荧光成像的细胞检测下限分别为5 × 10 3和5 × 10 4细胞。此外，使用表达TurboLuc的微环载体的流体动力学体内基因传递允许分析深层组织中随时间推移的发光信号。动物肝脏中的生物发光可监测30多天。总之，TurboLuc结合了生物发光和荧光的优势，允许从单细胞分析到活体全身生物发光成像的高灵敏度光学成像。TurboLuc融合报告蛋白的光学成像
Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 103 and 5 × 104 cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging. Optical imaging using TurboLuc fusion reporter protein
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影响因子：
11.2
作者：
Ray, Pritha;Tsien, Roger;Gambhir, Sanjiv Sam
通讯作者：
Gambhir, Sanjiv Sam
影响因子：
16.6
作者：
Shcherbakova, Daria M.;Subach, Oksana M.;Verkhusha, Vladislav V.
通讯作者：
Verkhusha, Vladislav V.
DOI：
10.1073/pnas.0704465104
发表时间：
2007-10-30
影响因子：
11.1
作者：
Naik, Snehal;Piwnica-Worms, David
通讯作者：
Piwnica-Worms, David
影响因子：
11.4
作者：
Rampelt, Heike;Kirstein-Miles, Janine;Bukau, Bernd
通讯作者：
Bukau, Bernd
DOI：
10.1007/978-1-61779-797-2_2
发表时间：
2012-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Hulit, James;Kedrin, Dmitriy;Segall, Jeffrey E
通讯作者：
Segall, Jeffrey E