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HSV1-TK/GFP/Fluc.

Authors

Zhang H1.

Source

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2008 Oct 21 [updated 2008 Dec 01].

Author information

1
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, Email: micad@ncbi.nlm.nih.gov

Excerpt

Reporter genes, also known as marker genes, possess a measurable phenotype distinguishable from the background of endogenous proteins (1). Several reporter genes express proteins that can generate signals for in vivo imaging, such as the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene, the green fluorescence protein (GFP) gene, and the firefly luciferase (Fluc) gene (2). The reporter gene is constructed with a “constitutive” promoter for continuous transcriptions or with an “inducible” promoter for controlled transcriptions (3). Both types of gene constructs have been used in the expression of exogenous genes and/or endogenous genes to monitor the levels of gene delivery and the efficiency in cell/tissue transduction in gene therapy. HSV1-TK/GFP/Fluc (TGL, Mw = 130 kDa) is a triple reporter protein used for multimodal in vivo imaging of gene expression (4) with single photon emission computed tomography (SPECT) or fluorescence and bioluminescence imaging. TGL is produced from the expression of a triple-fusion reporter gene (TGL gene) in which transcription and translation are processed through a single open reading frame. TGL consists of three fused protein subunits: HSV1-TK, GFP, and Fluc, where polylysine is used as a linker to connect the protein subunits and to maintain the molecular stability in the fused protein. Each subunit corresponds to a specific imaging modality. HSV1-TK is a homodimer composed of two 376-residue subunits (5), and it functions as a key enzyme in the pyrimidine-salvage pathway to catalyze the phosphorylation of thymidine (dT) to thymidine monophosphate (dTMP) in the presence of ATP and Mg2+. This feature has been used to design antiviral nucleoside analogs (i.e., ganciclovir) for antiviral therapy (5) and radiolabeled substrates such as radiolabeled 2’-fluoro-2’-deoxy-1-β-D-arabino-furanosyl-5-iodo-uracil (FIAU) for SPECT imaging (6). HSV can easily infect a variety of cells in that the expressed HSV1-TK can induce significant cytotoxicity in the presence of nucleoside analogs such as ganciclovir (7). For this reason, HSV1-TK gene is also widely used as a suicide gene in cancer treatment. 131I-Labeled FIAU is an active radiolabeled substrate of HSV1-TK that is detectable with SPECT (i.e., 131I emits gamma-ray at 364 keV (81% abundance) with a half-life time of 8.02 days, which can be detected with a gamma camera or SPECT. GFP is a fluorescent protein of 238 amino acids that emits a bright green fluorescence (λmax = 509 nm) when illuminated with a blue light (λmax = 395 nm) (8). The presence of the GFP subunit allows in vitro/in vivo fluorescence imaging and cell sorting with the fluorescence-activated cell sorting (FACS) technique. Fluc is an oxygenase (Mw = 62 kDa) extracted from Photinus pyralis (9). In the presence of adenosine triphosphate (ATP) and O2, Fluc oxidizes the heterocyclic substrate d-luciferin to oxyluciferin and emits light in the wavelength range of 400–620 nm (10). The Fluc subunit allows for planar whole-body bioluminescence imaging of gene-transduced cells.

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