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

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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HSV1-TK/GFP/Fluc

TGL
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: December 1, 2008.

Chemical name:HSV1-TK/GFP/Fluc
Abbreviated name:TGL
Synonym:TGL triple reporter
Agent category:Protein
Target:Other
Target category:Other – gene expression
Method of detection:Optical imaging, gamma imaging, SPECT
Source of signal/contrast:Green fluorescence protein (GFP), firefly luciferase, and 131I-FIAU
Activation:Yes
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
No structure is currently available in PubChem.

Background

[PubMed]

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.

Synthesis

[PubMed]

Ponomarev et al. described the preparation of the HSV1-TK/GFP/Fluc expression vector in multiple steps (4). First, a construct SFG-HSV1-TK/GFP was obtained (11). Plasmid pLTKRNL was digested with restriction enzyme BamHI to generate a 2.2-kb fragment that encoded the open reading frame of HSV1-TK. This fragment was ligated into plasmid pcDNA.1/Zeo to result in the 7.8-kb plasmid pTKZeo3.2; this ligation was followed by sequential digestions with BamHI and Xmal to produce a 1.2-kb TK fragment. At the same time, plasmid pEGFP-N1 was digested sequentially with BgIII and Xmal to generate a 4.7-kb DNA fragment containing the open reading frame of GFP. The 1.2-kb fragment was then ligated with the 4.7-kb fragment to form the 5.9-kb expression plasmid pTKGFP, in which the TK-GFP cDNA was under control of the cytomegalovirus immediately early (IE) promoter. After the ligation, the plasmid was cloned into a retroviral vector, the Moloney murine leukemia virus-based SFG vector, to yield SFG-TK-GFP. Second, two modified constructs, SFG-NES-HSV1-TK/GFP and SFG-Δ45-HSV1-TK/GFP, were obtained (6). To generate SFG-NES-HSV1-TK/GFP, the Xenopus cDNA for encoding the 20 amino acids in the nuclear export signal (NES) sequence of the mitogen-activated protein (MAP) kinase kinase (MAPKK) was inserted into the N-terminus of HSV1-TK/GFP between His-9 and Ala-10 using MluL. To generate SFG-Δ45-HSV1-TK/GFP, the Δ45-HSV1 cDNA lacking the first 45 amino acids (135 nucleotides) of the original HSV1-TK was amplified with selective primers and subsequently cloned between the Ncol sites of SFG-HSV1-TK/GFP. Third, the commercially available cDNA of fused Aequorea victoria eGFP and Fluc (eGFP/Fluc) was obtained from the plasmid pEGF/Pluc and subcloned into the retroviral vector SFG-Ntp to yield the construct SFG-GL (4). Finally, SFG-NES-HSV1-TK/GFP or SFG-Δ45-HSV1-TK/GFP was treated with Ncol and the fragment containing NES-HSV1-TK or Δ45-HSV1-TK was subcloned into the NcoI site of the SFG-GL to produce SFG-NES-HSV1-TK/GFP/Fluc (SFG-NES-TGL) or SFG-Δ45-HSV1-TK/GFP/Fluc (SFG-Δ45-TGL) (4). [131I]-FIAU with >97% radiochemical purity was prepared by the iododestannylation reaction using the tin precursor and carrier-free 131I.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Ponomarev et al. assessed the integrity of the fused reporter proteins with Western blot analysis (4). The molecular weight of the NES-TGL and Δ45-TGL proteins as determined with Western blot analysis matched the predicted molecular weights. These proteins were further examined with anti-HSV1-TK, anti-GFP, and anti-Fluc antibodies to demonstrate the coexistence of the triple subunits in the full reporter proteins. Ponomarev et al. then imaged the cytoplasmic localizations of the NES-TGL and Δ45-TGL reporter proteins with fluorescence microscopy (4). Human glioma U87 cells were transduced with the retroviral vectors SFG-NES-TGL or SFG-Δ45-TGL in vitro, followed by a bulk culture for 48 h. The obtained U87-NES-TGL cells or U87-Δ45-TGL cells were sorted for positive GFP expression using FACS with 488-nm excitation beam and 510-nm emission filter. The subcellular localization of NES-TGL and Δ45-TGL was visualized with fluorescence microscopy with similar excitation emission parameters. The GFP/Fluc signal appeared to be lower in the nucleus than in cytoplasm, but it was still detectable. Reporter gene expression in these transduced U87 cells was quantified with FACS, a bioluminescence assay, and a radiotracer [14C]-FIAU assay. The level of GFP fluorescence was found to be ~1.3 × 103 relative fluorescent units (RU) in the U87-NES-TGL cells and ~2.1 × 103 RU in the U87-Δ45-TGL cells. The influx rate constant Ki of 14C-labeled FIAU was ~1.4 media ml/min per g in the U87-NES-TGL cells and ~1.7 media ml/min per g in the U87-Δ45-TGL cells. The bioluminescence was ~2.8 × 103 relative bioluminescence unit (RLU) in the U87-NES-TGL cells and ~4.9 × 103 RLU in the U87-Δ45-TGL. The wild-type U87 cells were used as a control. Their measured fluorescence, influx rate, and bioluminescence were negligible.

Animal Studies

Rodents

[PubMed]

Ponomarev et al. used three imaging modalities to examine the expression of the triple reporter proteins in vivo (4). Nu/nu mice (20–25 g) were implanted subcutaneously with U87-NES-TGL cells on the right shoulder, with U87-Δ45-TGL cells on the left shoulder, and with wild-type U87 cells on the left flank. The tumors grew to an average size of 5.4 ± 2.1 mm and were imaged with whole-body fluorescence, bioluminescence, and gamma camera imaging. For bioluminescence imaging, mice were injected intravenously with 100 μl of D-luciferin solution at a dose of 150 mg/kg, and the images were acquired for 0.5–10 s. For the gamma camera imaging, mice received an intraperitoneal injection of 0.9% NaI solution (1 ml) one day before imaging to block the thyroid uptake of radioactive iodide. Then the mice were injected intravenously with [131I]-FIAU at a dose of 7.4 MBq per animal. The gamma camera images were collected 24 h after [131I]-FIAU injection to allow sufficient clearance of body background radioactivity. The GFP fluorescence was collected with a charge-coupled device (CCD) camera. The average level of gene expression in the implanted U87-NES-TGL tumor was detected as 707 ± 36 in fluorescence, 486 ± 47 × 106 photons/cm2 per sr in bioluminescence, and 0.47 ± 0.08% injected dose (ID)/g in [131I]-FIAU accumulation. The average level of gene expression in the implanted U87-Δ45-TGL tumor was detected as 740 ± 47 in fluorescence, 508 ± 36 × 106 photons/cm2 per sr in bioluminescence, and 0.86 ± 0.06% ID/g in [131I]-FIAU accumulation. As a control, the average level of gene expression in the implanted wild-type U87 cells was found at background level in fluorescence or bioluminescence imaging and at 0.03 ± 0.01% ID/g in [131I]-FIAU accumulation (the background level). After completion of the triple modality imaging, the mice were euthanized and the tissues were harvested for in vitro fluorescence imaging. Bright green fluorescent areas were found in the tumor tissues, demonstrating the presence of TGL labeling.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

References

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Massoud T.F., Singh A., Gambhir S.S. Noninvasive molecular neuroimaging using reporter genes: part I, principles revisited. AJNR Am J Neuroradiol. 2008;29(2):229–34. [PubMed: 18024575]
2.
Gross S., Piwnica-Worms D. Spying on cancer: molecular imaging in vivo with genetically encoded reporters. Cancer Cell. 2005;7(1):5–15. [PubMed: 15652745]
3.
Serganova I., Ponomarev V., Blasberg R. Human reporter genes: potential use in clinical studies. Nucl Med Biol. 2007;34(7):791–807. [PubMed: 17921031]
4.
Ponomarev V., Doubrovin M., Serganova I., Vider J., Shavrin A., Beresten T., Ivanova A., Ageyeva L., Tourkova V., Balatoni J., Bornmann W., Blasberg R., Gelovani Tjuvajev J. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur J Nucl Med Mol Imaging. 2004;31(5):740–51. [PubMed: 15014901]
5.
Wurth C., Thomas R.M., Folkers G., Scapozza L. Folding and self-assembly of herpes simplex virus type 1 thymidine kinase. J Mol Biol. 2001;313(3):657–70. [PubMed: 11676546]
6.
Ponomarev V., Doubrovin M., Serganova I., Beresten T., Vider J., Shavrin A., Ageyeva L., Balatoni J., Blasberg R., Tjuvajev J.G. Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of noninvasive molecular-genetic imaging. Neoplasia. 2003;5(3):245–54. [PMC free article: PMC1502405] [PubMed: 12869307]
7.
Kussmann-Gerber S., Kuonen O., Folkers G., Pilger B.D., Scapozza L. Drug resistance of herpes simplex virus type 1--structural considerations at the molecular level of the thymidine kinase. Eur J Biochem. 1998;255(2):472–81. [PubMed: 9716390]
8.
Chalfie M., Tu Y., Euskirchen G., Ward W.W., Prasher D.C. Green fluorescent protein as a marker for gene expression. Science. 1994;263(5148):802–5. [PubMed: 8303295]
9.
Conti E., Franks N.P., Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 1996;4(3):287–98. [PubMed: 8805533]
10.
Paulmurugan R., Gambhir S.S. Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal Chem. 2005;77(5):1295–302. [PMC free article: PMC4154832] [PubMed: 15732910]
11.
Jacobs A., Dubrovin M., Hewett J., Sena-Esteves M., Tan C.W., Slack M., Sadelain M., Breakefield X.O., Tjuvajev J.G. Functional coexpression of HSV-1 thymidine kinase and green fluorescent protein: implications for noninvasive imaging of transgene expression. Neoplasia. 1999;1(2):154–61. [PMC free article: PMC1508134] [PubMed: 10933050]
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