Triple Reporter for Bioluminescence, microPET (Positron Emission Tomography), and Fluorescence Imaging
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-[18F]fluoro-3-(hydroxymethyl) butyl guanine (18F-FHBG) microPET or 3H-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 IRESMGFP 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.
Schematic of pUB6-FLuc-mNLS-SR39TK.
(A) Bioluminescence imaging in vivo. Bioluminescence images obtained 24 hours following hydrodynamic delivery of control pUB6 or pUB6-FLuc-mNLS-SR39TK-IRES-MGFP plasmids. (B) Micro-positron emission tomography (microPET) in vivo. Coronal microPET images obtained 1 hour postinjection of 18F-FHBG in FVB mice 26 hours following hydrodynamic delivery of pUB6 control or pUB6-FLuc-mNLS-SR39TK-IRES-MGFP plasmids. Arrows point to the liver in each image. (C) Fluorescence imaging ex vivo. Green fluorescent protein expression levels from the livers of FVB mice collected 28 hours following hydrodynamic injection of pUB6 control (left) or pUB6-FLuc-mNLS-SR39TK-IRES-MGFP (right). Fluorescence images are superimposed over phase contrast images (340 original magnification).
Kesarwala AH, Prior JL, Sun J, Harpstrite SE, Sharma V, Piwnica-Worms D. Second-generation triple reporter for bioluminescence, microPET, and fluorescence imaging. Molec Imaging 2006; 5(4):465-74.