A method to rapidly and accurately compare the relative efficacies of non-invasive imaging reporter genes in a mouse model and its application to luciferase reporters.
A method to rapidly and accurately compare the relative efficacies of non-invasive imaging reporter genes in a mouse model and its application to luciferase reporters.
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DOI：
10.1007/s11307-011-0515-1
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发表时间：
2012-08
影响因子：
3.1
通讯作者：
Herschman, Harvey R.
中科院分区：
文献类型：
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作者：
Gil, Jose S.;Machado, Hidevaldo B.;Herschman, Harvey R.
关键词：
我们的目标是开发一种简单、定量、稳健的方法来比较成像报告基因在培养物和体内的功效。我们描述了腺病毒载体-肝脏转导程序，并比较了荧光素酶报告基因的功效。替代报告基因在常见的腺病毒载体中表达。体内使用的载体量基于细胞培养物滴定，确保每个载体使用相同的转导功效。成像后，使用定量实时 PCR 将体内和体外值标准化为肝载体转导。我们在细胞培养物和体内检测了标准萤火虫荧光素酶 (FLuc)、增强型萤火虫荧光素酶 (EFLuc)、荧光素酶 2 (Luc2)、人源化海肾荧光素酶 (hRLuc)、海肾荧光素酶 8.6-535 (RLuc8.6) 和膜结合高斯荧光素酶变体 (extGLuc)。我们观察到，与 FLuc 相比，EFLuc 和 Luc2 的生物发光信号增加了 100 倍以上，与 hRLuc 相比，RLuc8.6 的生物发光信号增加了 106 倍以上。在肝脏中未检测到 ExtGLuc。在某些情况下，我们的研究结果与先前比较这些报告基因得出的结论形成对比，并证明需要一种标准化方法来评估体内替代报告基因。我们的程序可以适用于利用替代成像方式（荧光、生物发光、MRI、SPECT、PET）的报告基因。
Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure, and compare the luciferase reporter efficacies. Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed a greater that 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc, and a greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes, and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET).
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影响因子：
3.6
作者：
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通讯作者：
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