Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain
Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain
复制标题
DOI：
10.1002/cmmi.1549
复制
发表时间：
2013-11-01
影响因子：
--
通讯作者：
Lowik, Clemens
中科院分区：
文献类型：
--
作者：
Mezzanotte, Laura;Aswendt, Markus;Lowik, Clemens
生物发光成像(BLI)已经成为对小型实验动物细胞进行光学跟踪的首选方法。然而，来自不同物种的荧光素酶的使用，取决于不同的底物和发射的不同波长，并没有针对敏感的神经成像进行优化。为了确定最合适的荧光素酶，本定量研究比较了荧光素酶Luc2、CBG99、PpyRE9和hRluc。以慢病毒载体为载体，将人胚胎肾(HEK-293)细胞和小鼠神经干细胞转化为四种荧光素酶之一，并与CopGFP一起表达。一个T2a多肽连接物促进了成像报告和流式细胞术细胞群比较之间的化学计量比表达。用细胞稀释法测定LUC2的最高BLI敏感性。然而，在体外，依赖于Coelenterazine h的hRluc信号明显超过了依赖d-荧光素的BLI。为了进行体内定量分析，将细胞移植到小鼠脑内，进行BLI，包括记录发射动力学和光谱特性。D-荧光素和Coelenterazine h的光动力学存在差异，Luc2和PpyRE9的发射光谱基本不变，而CBG99的发射光谱变为双相。最重要的是，光子发射按Luc2、CBG99、PpyRE9到hRluc的顺序递减。对不同荧光素酶联合应用于双色双底物神经成像的可行性进行了测试和讨论。这项研究首次对神经干细胞表达的不同荧光素酶进行了完整的定量比较。结果明确推荐Luc2作为体内神经成像的最佳荧光素酶选择。版权所有(C)2013 John Wiley&Sons，Ltd.
Bioluminescence imaging (BLI) has become the method of choice for optical tracking of cells in small laboratory animals. However, the use of luciferases from different species, depending on different substrates and emitting at distinct wavelengths, has not been optimized for sensitive neuroimaging. In order to identify the most suitable luciferase, this quantitative study compared the luciferases Luc2, CBG99, PpyRE9 and hRluc. Human embryonic kidney (HEK-293) cells and mouse neural stem cells were transduced by lentiviral vector-mediated transfer to express one of the four luciferases, together with copGFP. A T2A peptide linker promoted stoichiometric expression between both imaging reporters and the comparison of cell populations upon flow cytometry. Cell dilution series were used to determine highest BLI sensitivity in vitro for Luc2. However, Coelenterazine h-dependent hRluc signals clearly exceeded d-luciferin-dependent BLI in vitro. For the quantitative in vivo analysis, cells were transplanted into mouse brain and BLI was performed including the recording of emission kinetics and spectral characteristics. Differences in light kinetics were observed for d-luciferin vs Coelenterazine h. The emission spectra of Luc2 and PpyRE9 remained almost unchanged, while the emission spectrum of CBG99 became biphasic. Most importantly, photon emission decreased in the order of Luc2, CBG99, PpyRE9 to hRluc. The feasibility of combining different luciferases for dual color and dual substrate neuroimaging was tested and discussed. This investigation provides the first complete quantitative comparison of different luciferases expressed by neural stem cells. It results in a clear recommendation of Luc2 as the best luciferase selection for in vivo neuroimaging. Copyright (c) 2013 John Wiley & Sons, Ltd.