Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro

The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.     

Studying the biodistribution of positron emission tomography reporter probes in mice

Positron emission tomography (PET) reporter probes (PRPs) are used to detect PET reporter gene (PRG) expression in living subjects. This article details protocols for analyzing the biodistribution of a PRP used to detect herpes simplex virus 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk PRG expression. However, the methods described are generalizable to other beta- or gamma/positron-emitting probes. Accumulation of PRPs in animal tissues can be determined by counting PRP activity of isolated tissues, whereas digital whole-body autoradiography (DWBA) provides high-resolution images of PRP biodistribution in 5- to 45-microm tissue slices of killed research animals at a single time point. Biodistribution assay results may be obtained in less than a week after beginning the assay, and DWBA image acquisitions can take up to 3 months depending on the probe's radioisotope.     

Direct comparison of radiolabeled probes FMAU, FHBG, and FHPG as PET imaging agents for HSV1-tk expression in a human breast cancer model

2'-Deoxy-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FMAU), 9-(4-fluoro-3-hydroxy-methyl-butyl)guanine (FHBG) and 9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]-guanine (FHPG) have been evaluated in a human breast cancer model as potential radiotracers for PET imaging of HSV1-tk gene expression. In vitro accumulation of [14C]FMAU, [18F]FHBG, and [18F]FHPG in HSV1-tk-expressing cells was 14- to 16-fold (p <.001), 9- to 13-fold (p <.001), and 2- to 3-fold (p <.05) higher than tk-negative control cells, respectively, between 30 and 240 min. Accumulation of FMAU and FHBG in vector-transduced cells was 10- to 14-fold and 6- to 10-fold higher than wild-type cells, respectively. At 2 hr, uptake of [(14)C]FMAU in tkpositive cells was 6.3-fold and 60-fold higher than [18F]FHBG and [18F]FHPG, respectively. In vivo, tumor uptake of [14C]FMAU in HSV1-tk-expressing cells was 3.7-fold and 5.5-fold (p <.001) higher than tk-negative control cells at 1 and 2 hr, respectively. Tumor uptake of [18F]FHBG was 4.2-fold and 12.6-fold higher (p <.001) than tk-negative cells at the same time points. Incorporation of [14C]FMAU in tk-positive tumor was 18-fold and 24-fold higher (p <.001) than [18F]FHBG at 1 and 2 hr, respectively. Micro-PET images support the biodistribution results and indicate that both [18F]FMAU and [18F]FHBG are useful for imaging HSV1-tk expression in breast cancer. Although FMAU demonstrates higher total incorporation (%dose/g) in tumor tissue compared with the other tracers, FHBG is superior in terms of specific accumulation in transfected cells at later time points.     

Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-β-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.     

Multitracer positron emission tomographic imaging of exogenous gene expression mediated by a universal herpes simplex virus 1 amplicon vector

To develop efficient and safe gene therapy approaches, the herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET) using radiolabeled nucleoside analogues as specific TK substrates. Moreover, the gene encoding dopamine type 2 receptor (d2r) could be used as a PET marker gene using specific radiolabeled receptor binding compounds. Here we describe the quantitative colocalization of d2r and HSV-1-tk gene expression mediated from a universal HSV-1 amplicon vector in a subcutaneous human Gli36dEGFR glioma model by PET. The HSV-1 amplicon vector was constructed using a bicistronic gene cassette to contain (1) the d2r80A mutant, which is able to bind its ligand racloprid but unable to activate downstream signal transduction pathways, and (2) the tk39 mutant with enhanced enzymatic activity toward guanosine analogues fused to the green fluorescent protein gene (tk39gfp) serving as a marker gene in cell culture. After infection of human Gli36dEGFR glioma cells with the HSV-d2r80AIREStk39gfp (HSV-DITG) amplicon vector in cell culture, D2 receptor expression and its targeting to the cell surface were determined by Western blotting and immunolabeling. Vector application in vivo served for quantitative colocalization of d2r80A- and tk39gfp-derived PET signals employing the specific D2 receptor binding compound [(11)C]racloprid and the specific TK39 substrate 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine. Our results demonstrate that for the range of gene expression studied in vivo, both enzymatic and receptor binding assays give comparable quantitative information on the level of vector-mediated gene expression in vivo. The d2r80A in combination with a specific binding compound passing the intact blood-brain barrier might be an alternative marker gene for the noninvasive assessment of vector-mediated gene expression in the brain using PET.     

Molecular PET imaging of HSV1-tk reporter gene expression using [18F]FEAU

Non-invasive imaging of transgene expression requires the appropriate combination of a reporter gene and a reporter probe. [18F]FEAU positron emission tomography (PET) is used for the assessment of herpes simplex virus type-1 thymidine kinase gene expression. Hybrid AAV phage (termed AAVP) can be adapted to transduce mammalian cells by targeting to a specific receptor. We evaluated a targeted AAVP vector using [18F]FEAU PET. This protocol describes [18F]FEAU production and dosing, micro-PET imaging and image analysis. 2-Deoxy-2-trifluoromethanesulfonyl-1,3,5-tri-O-benzoyl-alpha-D-ribofuranose is radio-fluorinated, converted into its 1-bromo derivative and coupled with protected 5-ethyl uracil. The coupled product is hydrolyzed and purified using HPLC. Tumor-bearing animals targeted with either retroviral or AAVP vectors are anesthetized and injected with [18F]FEAU (0.1 mCi per mouse); this is followed 2 h after injection by imaging on a micro-PET. Production of [18F]FEAU requires approximately 3.5 h from the end of bombardment. PET imaging studies require 2-3 h (depending on the number of animals) after synthesis of [18F]FEAU.     

Imaging expression of cytosine deaminase-herpes virus thymidine kinase fusion gene (CD/TK) expression with [124I]FIAU and PET

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)-herpes simplex virus type 1 thymidine kinase (HSV1-tk) fusion gene (CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.     

Multimodal imaging of neural progenitor cell fate in rodents

For clinical application of stem cell-based therapies, noninvasive detection of applied stem cells is of high importance. We report on the feasibility of detecting implanted neural progenitor cells (NPCs) noninvasively and follow their fate and functional status by sequential multimodal molecular imaging and reporter gene technology. We investigated C17.2 cells stably expressing herpes simplex virus type 1-thymidine kinase (HSV-1-tk) and green fluorescent protein (gfp) (C17.2-tkIRESgfp = C17.2-TIG) or HSV-1-tk, gfp, and firefly luciferase (luc) (C17.2-lucIREStkgfp = C17.2-LITG) and determined the detection sensitivity of positron emission tomography (PET) and bioluminescence imaging (BLI) for these cells in culture and in vivo in subcutaneous and intracranial glioma models. In addition, PET and BLI were used to further investigate and follow the fate of implanted C17.2-LITG cells in an intracranial glioma model. We show that both imaging modalities are sensitive in detecting reporter gene expressing NPCs; however, PET, by the use of 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG), detects NPCs only at sites of disrupted blood-brain barrier. Furthermore, both imaging modalities can be used to detect stem cell fate and migration and indicate excessive proliferation and aberrant migration. In conclusion, multimodal imaging can be used for longitudinal noninvasive monitoring of grafted NPCs in rodents.     

Molecular Imaging of CXCL12 Promoter-driven HSV1-TK Reporter Gene Expression

The C-X-C motif chemokine 12 (CXCL12, SDF1a) and its receptor, CXCR4, play a fundamental role in several biological processes, including hematopoiesis, cardiogenesis, cancer progression, and stem cell migration. Noninvasive monitoring of CXCL12 is highly desirable for optimizing strategies that combine mobilization of therapeutic cells to combat cancer or to assist in cardiac tissue repair after myocardial infarction. Here, we report on an MRI reporter gene system for directly monitoring CXCL12 expression in vivo. Glioma cells and human adipose-derived stem cells (hADSC) were transduced with the herpes simplex virus type-1-thymidine kinase (HSV1- tk) reporter gene expressed under the CXCL12 promoter. HSV1-tk expression resulted in accumulation of the PET tracer [¹²⁵I]FIAU in vitro and in vivo and induced cell death after ganciclovir treatment. Furthermore, the results show that conditional expression of the reporter gene can be induced by hypoxia in transduced cells. Transduced hADSC were incubated with the CEST MRI probe 5-methyl-5, 6- dihydrothymidine (5-MDHT) and transplanted into swine heart. Transplanted cells were clearly visible on Chemical Exchange Saturation Transfer (CEST) MRI using a 3T clinical scanner. Therefore, we conclude that it is possible to image CXCL12 expression with MRI in a large animal model, opening up a possible route to clinical translation.     

Imaging long-term fate of intramyocardially implanted mesenchymal stem cells in a porcine myocardial infarction model

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.     

Synthesis and characterization of a C6 nucleoside analogue for the in vivo imaging of the gene expression of herpes simplex virus type-1 thymidine kinase (HSV1 TK)

The synthesis and biological evaluation of '6-(1,3-dihydroxyisobutyl)thymine' (DHBT; 1), which corresponds to 6-[3-hydroxy-2-(hydroxymethyl)propyl]-5-methylpyrimidine-2,4(1H,3H)-dione, is reported. DHBT (1) was designed as a new substrate for herpes simplex virus type-1 thymidine kinase (HSV1 TK). The compound was found to be exclusively phosphorylated by HSV1 TK, and to exhibit good binding affinity (Ki = 35.3+/-1.3 microM). Cell-proliferation assays with HSV1-TK-transduced human osteosarcoma cells (143B-TK+-HSV1-WT) and with both human-thymidine-kinase-1-negative (143B-TK-) and non-transduced parental (MG-63) cells indicate that 1 is less cytotoxic than the standard drug Ganciclovir. Thus, DHBT (1) represents a promising precursor of a nontoxic reporter probe for the monitoring of HSV1 TK gene expression by means of positron-emission tomography (PET).     

18F]FHPG positron emission tomography for detection of herpes simplex virus (HSV) in experimental HSV encephalitis

Herpes simplex virus type 1 (HSV-1) is one of the most common causes of sporadic encephalitis. The initial clinical course of HSV encephalitis (HSE) is highly variable, and the infection may be rapidly fatal. For effective treatment with antiviral medication, an early diagnosis of HSE is crucial. Subtle brain infections with HSV may be causally related to neuropsychiatric disorders such as Alzheimer's dementia. We investigated the feasibility of a noninvasive positron emission tomography (PET) imaging technique using [(18)F]FHPG as a tracer for the detection of HSE. For this purpose, rats received HSV-1 (infected group) or phosphate-buffered saline (control group) by intranasal application, and dynamic PET scans were acquired. In addition, the distribution of tracer accumulation in specific brain areas was studied with phosphor storage imaging. The PET images revealed that the overall brain uptake of [(18)F]FHPG was significantly higher for the infected group than for control animals. Phosphor storage images showed an enhanced accumulation of [(18)F]FHPG in regions known to be affected after intranasal infection with HSV. High-performance liquid chromatography metabolite analysis showed phosphorylated metabolites of [(18)F]FHPG in infected brains, proving that the increased [(18)F]FHPG uptake in infected brains was due to HSV thymidine kinase-mediated trapping. Freeze lesion experiments showed that damage to the blood-brain barrier could in principle induce elevated [(18)F]FHPG uptake, but this nonspecific tracer uptake could easily be discriminated from HSE-derived uptake by differences in the tracer kinetics. Our results show that [(18)F]FHPG PET is a promising tool for the detection of HSV encephalitis.     

Second-generation triple reporter for bioluminescence, micro-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-[(18)F]fluoro-3-(hydroxymethyl) butyl guanine ((18)F-FHBG) microPET or (3)H-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLuc-mNLS-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 (18)F-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.     

In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.     

Evaluation of 2'-deoxy-2'-flouro-5-methyl-1-beta-D-arabinofuranosyluracil as a potential gene imaging agent for HSV-tk expression in vivo

2'-Deoxy-2'-flouro-5-methyl-1-beta-D-arabinofuranosyluracil (FMAU) has been evaluated in HT-29 cells as a potential positron emission tomography (PET) radiotracer for imaging HSV-tk gene expression in vivo. In vitro experiments demonstrate that the accumulation of [14C]-FMAU in HSV-tk-expressing cells is 2.4-fold (p < .02), 4.0-fold (p < .001), and 5.3-fold (p < .001) higher than the wild-type cells at 1, 3, and 5 hr, respectively. In vivo studies revealed that the tumor uptake in HSV-tk-expressing cells was 2.3-fold (p < .001), 3.0-fold (p < .001), and 5.5-fold (p < .001) higher than the control cells at 1, 2, and 5 hr, respectively. FMAU was found to be more sensitive compared to our earlier studies using 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) and 9-(4-[18F]-fluoro-3-hydroxy-methylbutyl)guanine ([18F]-FHBG) in the same cell lines, although, the specificity was less than FHBG. These results suggest that while FMAU labeled with PET isotopes may be useful for imaging HSV-tk-expressing tumors in vivo, multitracer studies across additional tumor models are necessary in order to identify an optimal PET radiotracer.     

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [¹⁸F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers.     

I型单纯疱疹病毒胸苷激酶真核表达载体的构建及其表达鉴定

为构建单纯疱疹病毒I型胸苷激酶(HSV1TK)的真核表达载体pcDNA3.1-EGFP/HSV1TK,鉴定其在真核细胞中的表达和功能.以pORF-HSV1TK为模板,PCR扩增的目的基因HSV1TK片段与pMD18-T载体相连接构建重组克隆pMD18-T/HSV1TK.再双酶切出HSV1TK片段,插入pcDNA3.1-EGFP多克隆位点,构建pcDNA3.1-EGFP/ HSV1TK真核表达载体并进行酶切、测序鉴定[1].分别用荧光显微镜观察和RT-PCR方法检测脂质体介导pcDNA3.1-EGFP/ HSV1TK在卵巢癌细胞SKOV3的表达;分别用MTT法和光镜检测胸苷激酶/丙氧鸟苷(HSV1TK/GCV)系统对SKOV3体外杀伤作用及旁观者效应.结果表明,重组载体酶切鉴定结果与预期结果一致,基因序列与GenBank上报道的HSV1TK基因序列完全一致.荧光显微镜观察转染后的细胞发出绿色荧光;RT-PCR结果表明HSV1TK基因能在SKOV3内有效表达.MTT和光镜结果显示转染HSV1TK基因的SKOV3细胞,加入前体药物丙氧鸟苷(GCV)处理后对其有明显的杀伤作用和旁观者效应.成功构建的真核表达载体pcDNA3.1-EGFP/ HSV1TK能在SKOV3细胞中稳定表达,且HSV1TK对卵巢癌细胞株SKOV3体外有强大的杀伤作用和旁观者效应.     

Novel radiosynthesis of PET HSV-tk gene reporter probes [18F]FHPG and [18F]FHBG employing dual Sep-Pak SPE techniques

Positron emission tomography (PET) herpes simplex virus thymidine kinase (HSV-tk) gene reporter probes 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) and 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) were prepared by nucleophilic substitution of the appropriate tosylated precursors with [(18)F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified dual Silica Sep-Pak solid-phase extraction (SPE) method in 15-30% radiochemical yield.