Second-generation triple reporter for bioluminescence, micro-positron emission tomography, and fluorescence imaging
Second-generation triple reporter for bioluminescence, micro-positron emission tomography, and fluorescence imaging
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DOI：
10.2310/7290.2006.00024
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发表时间：
2006-10-01
影响因子：
2.8
通讯作者：
Piwnica-Worms, David
中科院分区：
文献类型：
--
作者：
Kesarwala, Aparna H.;Prior, Julie L.;Piwnica-Worms, David
生物发光、正电子发射断层扫描（PET）和荧光模式目前可用于体内非侵入性成像，每一种都有其自身的优点。为了利用各自的综合优势并促进多模态成像，我们设计了一种双报告子构建体，其中萤火虫荧光素酶（FLuc）和12个氨基酸的非结构性连接子与突变型单纯疱疹病毒胸苷激酶（mNLS-SR 39 TK）的N-末端融合，该酶为正电子发射断层扫描（PET）动力学增强。此外，开发了三重报告构建体，其中将怪物绿色荧光蛋白（MGFP）（一种最近可获得的增强型荧光蛋白）引入融合载体的内部核糖体进入位点（IRES）下游，以允许通过荧光显微镜或流式细胞术进行分析，而不损害上游融合组分的特异性活性。用冷却的电荷耦合器件照相机测量FLuc生物发光，并通过9-[4[（18）F]-氟-3-（羟甲基）丁基鸟嘌呤（F-18-FHBG）microPET或H-3-阿昔洛韦净积累测量mNLS-SR 39 TK活性。重要的是，用FLuc-mNLS-SR 39 TK-IRES-MGFP三重报告基因瞬时转染的HeLa细胞保留了FLucmNLS-SR 39 TK杂酶和单个未融合酶的相同特异性活性，而蛋白质半衰期没有变化。IRES-MGFP的存在适度降低了上游杂蛋白表达。在活小鼠中，与用对照质粒处理的小鼠相比，泛素启动子驱动的FLuc-mNLS-SR 39 TK-IRES-MGFP质粒的体细胞基因转移显示肝脏光子通量增加> 1，000倍，并且通过microPET显示18F-FHBG的肝脏保留增加> 2倍。通过标准共聚焦显微镜容易观察到多焦肝细胞荧光。这种第二代三重报告基因结合了增强的成分，能够对细胞和活体动物进行生物发光、PET和荧光成像。
Bioluminescence, positron emission tomography (PET), and fluorescence modalities are currently available for noninvasive imaging in vivo, each with its own merits. To exploit the combined strengths of each and facilitate multimodality imaging, we engineered a dual-reporter construct in which firefly luciferase (FLuc) and a 12-amino acid nonstructural linker were fused in frame to the N-terminus of a mutant herpes simplex virus thymidine kinase (mNLS-SR39TK) kinetically enhanced for positron emission tomography (PET). Furthermore, a triple-reporter construct was developed in which monster green fluorescent protein (MGFP), a recently available enhanced fluorescent protein, was introduced into the fusion vector downstream of an internal ribosome entry site (IRES) to allow analysis by fluorescence microscopy or flow cytometry without compromising the specific activities of the upstream fusion components. FLuc bioluminescence was measured with a cooled charge-coupled device camera and mNLS-SR39TK activity by 9-[4[(18)FIfluoro-3-(hydroxymethyl) butyl guanine (F-18-FHBG) microPET or H-3-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLucmNLS-SR39TK heteroenzyme and the individual unfused enzymes with no change in protein half-lives. The presence of the IRES-MGFP modestly decreased upstream heteroprotein expression. In living mice, somatic gene transfer of a ubiquitin promoter-driven FLuc-mNLS-SR39TK-IRES-MGFP plasmid showed a > 1,000-fold increase in liver photon flux and a > 2-fold increase in liver retention of 18F-FHBG by microPET compared with mice treated with control plasmid. Multifocal hepatocellular fluorescence was readily observed by standard confocal microscopy. This second-generation triple reporter incorporating enhanced components enables bioluminescence, PET, and fluorescence imaging of cells and living animals.