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Sanjiv S Gambhir - One of the best experts on this subject based on the ideXlab platform.
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synthesis and characterization of 9 4 18f fluoro 3 hydroxymethyl butyl 2 phenylthio 6 oxopurine as a novel pet agent for mutant herpes simplex virus type 1 thymidine kinase Reporter Gene imaging
Molecular Imaging and Biology, 2020Co-Authors: Takeshi Fuchigami, Mohammad Namavari, Tom Haywood, Gayatri Gowrishankar, David Anders, Mirwais Wardak, Sanjiv S GambhirAbstract:[18F]FHBG has been used as a positron emission tomography (PET) imaging tracer for the monitoring of herpes simplex virus type 1 thymidine kinase (HSV1-tk), a Reporter Gene for cell and Gene therapy in humans. However, this tracer shows inadequate blood-brain barrier (BBB) penetration and, therefore, would be limited for accurate quantification of Reporter Gene expression in the brain. Here, we report the synthesis and evaluation of 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)-2(phenylthio)-6-oxopurine ([18F]FHBT) as a new PET tracer for imaging Reporter Gene expression of HSV1-tk and its mutant HSV1-sr39tk, with the aim of improved BBB penetration. [18F]FHBT was prepared by using a tosylate precursor and [18F]KF. The cellular uptake of [18F]FHBT was performed in HSV1-sr39tk-positive (+) or HSV1-sr39tk-negative (−) MDA-MB-231 breast cancer cells. The specificity of [18F]FHBT to assess HSV1-sr39tk expression was evaluated by in vitro blocking studies using 1 mM of ganciclovir (GCV). Penetration of [18F]FHBT and [18F]FHBG across the BBB was assessed by dynamic PET imaging studies in normal mice. The tosylate precursor reacted with [18F]KF using Kryptofix2.2.2 followed by deprotection to give [18F]FHBT in 10 % radiochemical yield (decay-corrected). The uptake of [18F]FHBT in HSV1-sr39tk (+) cells was significantly higher than that of HSV1-sr39tk (−) cells. In the presence of GCV (1 mM), the uptake of [18F]FHBT was significantly decreased, indicating that [18F]FHBT serves as a selective substrate of HSV1-sr39TK. PET images and time-activity curves of [18F]FHBT in the brain regions showed similar initial brain uptakes (~ 12.75 min) as [18F]FHBG (P > 0.855). Slower washout of [18F]FHBT was observed at the later time points (17.75 − 57.75 min, P > 0.207). Although [18F]FHBT showed no statistically significant improvement of BBB permeability compared with [18F]FHBG, we have demonstrated that the 2-(phenylthio)-6-oxopurine backbone can serve as a novel scaffold for developing HSV1-tk/HSV1-sr39tk Reporter Gene imaging agents for additional research in the future.
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uptake kinetics and biodistribution of 14c d luciferin a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction impact on bioluminescence based Reporter Gene imaging
European Journal of Nuclear Medicine and Molecular Imaging, 2008Co-Authors: F Berger, Ramasamy Paulmurugan, Srabani Bhaumik, Sanjiv S GambhirAbstract:Purpose Firefly luciferase catalyzes the oxidative decarboxylation of d-luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase Reporter Gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated.
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imaging tri fusion multimodality Reporter Gene expression in living subjects
Cancer Research, 2004Co-Authors: Abhijit De, Roger Y Tsien, Sanjiv S GambhirAbstract:Imaging Reporter Gene expression in living subjects with various imaging modalities is a rapidly accelerating area of research. Applications of these technologies to cancer research, Gene therapy, and transgenic models are rapidly expanding. We report construction and testing of several triple fusion Reporter Genes compatible with bioluminescence, fluorescence and positron emission tomography (PET) imaging. A triple fusion Reporter vector harboring a bioluminescence synthetic Renilla luciferase ( hrl ) Reporter Gene, a Reporter Gene encoding the monomeric red fluorescence protein ( mrfp1 ), and a mutant herpes simplex virus type 1 sr39 thymidine kinase [ HSV1-truncated sr39tk ( ttk ); a PET Reporter Gene] was found to preserve the most activity for each protein component and was therefore investigated in detail. After validating the activities of all three proteins encoded by the fusion Gene in cell culture, we imaged living mice bearing 293T cells transiently expressing the hrl-mrfp-ttk vector by microPET and using a highly sensitive cooled charge-coupled device camera compatible with both bioluminescence and fluorescence imaging. A lentiviral vector carrying the triple fusion Reporter Gene was constructed and used to isolate stable expressers by fluorescence-activated cell sorting. These stable 293T cells were further used to show good correlation (R2 ∼0.74–0.85) of signal from each component by imaging tumor xenografts in living mice with all three modalities. Furthermore, metastases of a human melanoma cell line (A375M) stably expressing the triple fusion were imaged by microPET and optical technologies over a 40–50-day time period in living mice. Imaging of Reporter Gene expression from single cells to living animals with the help of a single tri-fusion Reporter Gene will have the potential to accelerate translational cancer research.
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optical bioluminescence and positron emission tomography imaging of a novel fusion Reporter Gene in tumor xenografts of living mice
Cancer Research, 2003Co-Authors: Anna M Wu, Sanjiv S GambhirAbstract:Noninvasive imaging of Reporter Gene expression using various imaging modalitiesis playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and Gene therapy. In this study, a novel Reporter vector was constructed encoding a fusion protein comprised of a mutant herpes simplex virus type 1 thymidine kinase ( HSV1-sr39tk ) ( tk ), a positron emission tomography (PET) Reporter Gene, and renilla luciferase ( rl ), a bioluminescence optical Reporter Gene joined by a 20 amino acid long spacer sequence. We validated the activity of the two enzymes encoded by the fusion protein ( tk 20 rl ) in cell culture. Then, tumors stably expressing the tk 20 rl fusion Gene were imaged both by microPET and optically using a cooled charge coupled device camera in xenograft-bearing living mice. Using a single fusion Reporter (PET/optical) Gene should accelerate the validation of Reporter Gene approaches developed in cell culture for translation into preclinical and clinical models.
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monitoring adenoviral dna delivery using a mutant herpes simplex virus type 1 thymidine kinase Gene as a pet Reporter Gene
Gene Therapy, 2002Co-Authors: Qianwa Liang, Sanjiv S Gambhir, Jorge R. Barrio, Michael E. Phelps, Khoi Nguyen, Nagichettiar Satyamurthy, Harvey R. HerschmanAbstract:Current Gene therapy protocols often suffer from an inability to monitor the site, level and persistence of Gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a Reporter Gene for in vivo imaging of Reporter Gene expression. The presence of the HSV1-tk Reporter Gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET Reporter Gene system, both alternative substrates and mutations in the HSV1-tk Gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[18F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET Reporter Gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.
Jorge R. Barrio - One of the best experts on this subject based on the ideXlab platform.
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monitoring adenoviral dna delivery using a mutant herpes simplex virus type 1 thymidine kinase Gene as a pet Reporter Gene
Gene Therapy, 2002Co-Authors: Qianwa Liang, Sanjiv S Gambhir, Jorge R. Barrio, Michael E. Phelps, Khoi Nguyen, Nagichettiar Satyamurthy, Harvey R. HerschmanAbstract:Current Gene therapy protocols often suffer from an inability to monitor the site, level and persistence of Gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a Reporter Gene for in vivo imaging of Reporter Gene expression. The presence of the HSV1-tk Reporter Gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET Reporter Gene system, both alternative substrates and mutations in the HSV1-tk Gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[18F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET Reporter Gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.
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A mutant herpes simplex virus type 1 thymidine kinase Reporter Gene shows improved sensitivity for imaging Reporter Gene expression with positron emission tomography
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Sanjiv S Gambhir, Eileen Bauer, Margaret E. Black, Qianwa Liang, Mark S. Kokoris, Jorge R. Barrio, Meera Iyer, Mohammad Namavari, Michael E. Phelps, Harvey R. HerschmanAbstract:We are developing assays for noninvasive, quantitative imaging of Reporter Genes with positron emission tomography (PET), for application both in animal models and in human Gene therapy. We report here a method to improve the detection of lower levels of PET Reporter Gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET Reporter Gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8-3H]penciclovir ([8-3H]PCV), and 8-[18F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of ≈2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET Reporter Gene and FPCV as a PET Reporter probe results in significantly enhanced sensitivity for imaging Reporter Gene expression in vivo.
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imaging of adenoviral directed herpes simplex virus type 1 thymidine kinase Reporter Gene expression in mice with radiolabeled ganciclovir
The Journal of Nuclear Medicine, 1998Co-Authors: Sanjiv S Gambhir, Eileen Bauer, Jorge R. Barrio, Meera Iyer, Mohammad Namavari, Nagichettiar Satyamurthy, Lily Wu, Capella Parrish, Duncan C Maclaren, Ali R BorgheiAbstract:We are developing procedures to repeatedly and noninvasively image the expression of transplanted Reporter Genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase Gene (HSV1-tk) as a Reporter Gene and [8- 14 C]-ganciclovir as a Reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8- 14 C]-ganciclovir, As a result, specific accumulation of phosphorylated [8- 14 C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk Gene. Methods: An adenoviral vector was constructed carrying the HSV1-tk Gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8- 3 H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8- 14 C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies. Results: Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a Reporter Gene and [8- 14 C]-ganciclovir as an imaging Reporter probe. A good correlation (r 2 = 0.86) between the [8- 14 C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8- 18 F]-fluoganciclovir and microPET imaging supports further investigation of [8- 18 F]-fluoroganciclovir as a PET Reporter probe for imaging HSV1-tk Gene expression. Conclusion: These results demonstrate the feasibility of using [8- 14 C]-ganciclovir as a Reporter probe for the HSV1-tk Reporter Gene, using an in vivo adenoviral mediated Gene delivery system in a murine model. The results form the foundation for further investigation of [8- 18 F]-fluoroganciclovir for noninvasive and repeated imaging of Gene expression with PET.
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imaging of adenoviral directed herpes simplex virus type 1 thymidine kinase Reporter Gene expression in mice with radiolabeled ganciclovir
The Journal of Nuclear Medicine, 1998Co-Authors: Sanjiv S Gambhir, Eileen Bauer, Jorge R. Barrio, Meera Iyer, Mohammad Namavari, Nagichettiar Satyamurthy, Capella Parrish, Duncan C Maclaren, Ali R Borghei, Leeta A GreenAbstract:We are developing procedures to repeatedly and noninvasively image the expression of transplanted Reporter Genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase Gene (HSV1-tk) as a Reporter Gene and [8- 14 C]-ganciclovir as a Reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8- 14 C]-ganciclovir, As a result, specific accumulation of phosphorylated [8- 14 C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk Gene. Methods: An adenoviral vector was constructed carrying the HSV1-tk Gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8- 3 H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8- 14 C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies. Results: Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a Reporter Gene and [8- 14 C]-ganciclovir as an imaging Reporter probe. A good correlation (r 2 = 0.86) between the [8- 14 C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8- 18 F]-fluoganciclovir and microPET imaging supports further investigation of [8- 18 F]-fluoroganciclovir as a PET Reporter probe for imaging HSV1-tk Gene expression. Conclusion: These results demonstrate the feasibility of using [8- 14 C]-ganciclovir as a Reporter probe for the HSV1-tk Reporter Gene, using an in vivo adenoviral mediated Gene delivery system in a murine model. The results form the foundation for further investigation of [8- 18 F]-fluoroganciclovir for noninvasive and repeated imaging of Gene expression with PET.
Harvey R. Herschman - One of the best experts on this subject based on the ideXlab platform.
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monitoring adenoviral dna delivery using a mutant herpes simplex virus type 1 thymidine kinase Gene as a pet Reporter Gene
Gene Therapy, 2002Co-Authors: Qianwa Liang, Sanjiv S Gambhir, Jorge R. Barrio, Michael E. Phelps, Khoi Nguyen, Nagichettiar Satyamurthy, Harvey R. HerschmanAbstract:Current Gene therapy protocols often suffer from an inability to monitor the site, level and persistence of Gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a Reporter Gene for in vivo imaging of Reporter Gene expression. The presence of the HSV1-tk Reporter Gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET Reporter Gene system, both alternative substrates and mutations in the HSV1-tk Gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[18F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET Reporter Gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.
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A mutant herpes simplex virus type 1 thymidine kinase Reporter Gene shows improved sensitivity for imaging Reporter Gene expression with positron emission tomography
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Sanjiv S Gambhir, Eileen Bauer, Margaret E. Black, Qianwa Liang, Mark S. Kokoris, Jorge R. Barrio, Meera Iyer, Mohammad Namavari, Michael E. Phelps, Harvey R. HerschmanAbstract:We are developing assays for noninvasive, quantitative imaging of Reporter Genes with positron emission tomography (PET), for application both in animal models and in human Gene therapy. We report here a method to improve the detection of lower levels of PET Reporter Gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET Reporter Gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8-3H]penciclovir ([8-3H]PCV), and 8-[18F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of ≈2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET Reporter Gene and FPCV as a PET Reporter probe results in significantly enhanced sensitivity for imaging Reporter Gene expression in vivo.
Mohammad Namavari - One of the best experts on this subject based on the ideXlab platform.
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synthesis and characterization of 9 4 18f fluoro 3 hydroxymethyl butyl 2 phenylthio 6 oxopurine as a novel pet agent for mutant herpes simplex virus type 1 thymidine kinase Reporter Gene imaging
Molecular Imaging and Biology, 2020Co-Authors: Takeshi Fuchigami, Mohammad Namavari, Tom Haywood, Gayatri Gowrishankar, David Anders, Mirwais Wardak, Sanjiv S GambhirAbstract:[18F]FHBG has been used as a positron emission tomography (PET) imaging tracer for the monitoring of herpes simplex virus type 1 thymidine kinase (HSV1-tk), a Reporter Gene for cell and Gene therapy in humans. However, this tracer shows inadequate blood-brain barrier (BBB) penetration and, therefore, would be limited for accurate quantification of Reporter Gene expression in the brain. Here, we report the synthesis and evaluation of 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)-2(phenylthio)-6-oxopurine ([18F]FHBT) as a new PET tracer for imaging Reporter Gene expression of HSV1-tk and its mutant HSV1-sr39tk, with the aim of improved BBB penetration. [18F]FHBT was prepared by using a tosylate precursor and [18F]KF. The cellular uptake of [18F]FHBT was performed in HSV1-sr39tk-positive (+) or HSV1-sr39tk-negative (−) MDA-MB-231 breast cancer cells. The specificity of [18F]FHBT to assess HSV1-sr39tk expression was evaluated by in vitro blocking studies using 1 mM of ganciclovir (GCV). Penetration of [18F]FHBT and [18F]FHBG across the BBB was assessed by dynamic PET imaging studies in normal mice. The tosylate precursor reacted with [18F]KF using Kryptofix2.2.2 followed by deprotection to give [18F]FHBT in 10 % radiochemical yield (decay-corrected). The uptake of [18F]FHBT in HSV1-sr39tk (+) cells was significantly higher than that of HSV1-sr39tk (−) cells. In the presence of GCV (1 mM), the uptake of [18F]FHBT was significantly decreased, indicating that [18F]FHBT serves as a selective substrate of HSV1-sr39TK. PET images and time-activity curves of [18F]FHBT in the brain regions showed similar initial brain uptakes (~ 12.75 min) as [18F]FHBG (P > 0.855). Slower washout of [18F]FHBT was observed at the later time points (17.75 − 57.75 min, P > 0.207). Although [18F]FHBT showed no statistically significant improvement of BBB permeability compared with [18F]FHBG, we have demonstrated that the 2-(phenylthio)-6-oxopurine backbone can serve as a novel scaffold for developing HSV1-tk/HSV1-sr39tk Reporter Gene imaging agents for additional research in the future.
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A mutant herpes simplex virus type 1 thymidine kinase Reporter Gene shows improved sensitivity for imaging Reporter Gene expression with positron emission tomography
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Sanjiv S Gambhir, Eileen Bauer, Margaret E. Black, Qianwa Liang, Mark S. Kokoris, Jorge R. Barrio, Meera Iyer, Mohammad Namavari, Michael E. Phelps, Harvey R. HerschmanAbstract:We are developing assays for noninvasive, quantitative imaging of Reporter Genes with positron emission tomography (PET), for application both in animal models and in human Gene therapy. We report here a method to improve the detection of lower levels of PET Reporter Gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET Reporter Gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8-3H]penciclovir ([8-3H]PCV), and 8-[18F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of ≈2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET Reporter Gene and FPCV as a PET Reporter probe results in significantly enhanced sensitivity for imaging Reporter Gene expression in vivo.
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imaging of adenoviral directed herpes simplex virus type 1 thymidine kinase Reporter Gene expression in mice with radiolabeled ganciclovir
The Journal of Nuclear Medicine, 1998Co-Authors: Sanjiv S Gambhir, Eileen Bauer, Jorge R. Barrio, Meera Iyer, Mohammad Namavari, Nagichettiar Satyamurthy, Lily Wu, Capella Parrish, Duncan C Maclaren, Ali R BorgheiAbstract:We are developing procedures to repeatedly and noninvasively image the expression of transplanted Reporter Genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase Gene (HSV1-tk) as a Reporter Gene and [8- 14 C]-ganciclovir as a Reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8- 14 C]-ganciclovir, As a result, specific accumulation of phosphorylated [8- 14 C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk Gene. Methods: An adenoviral vector was constructed carrying the HSV1-tk Gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8- 3 H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8- 14 C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies. Results: Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a Reporter Gene and [8- 14 C]-ganciclovir as an imaging Reporter probe. A good correlation (r 2 = 0.86) between the [8- 14 C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8- 18 F]-fluoganciclovir and microPET imaging supports further investigation of [8- 18 F]-fluoroganciclovir as a PET Reporter probe for imaging HSV1-tk Gene expression. Conclusion: These results demonstrate the feasibility of using [8- 14 C]-ganciclovir as a Reporter probe for the HSV1-tk Reporter Gene, using an in vivo adenoviral mediated Gene delivery system in a murine model. The results form the foundation for further investigation of [8- 18 F]-fluoroganciclovir for noninvasive and repeated imaging of Gene expression with PET.
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imaging of adenoviral directed herpes simplex virus type 1 thymidine kinase Reporter Gene expression in mice with radiolabeled ganciclovir
The Journal of Nuclear Medicine, 1998Co-Authors: Sanjiv S Gambhir, Eileen Bauer, Jorge R. Barrio, Meera Iyer, Mohammad Namavari, Nagichettiar Satyamurthy, Capella Parrish, Duncan C Maclaren, Ali R Borghei, Leeta A GreenAbstract:We are developing procedures to repeatedly and noninvasively image the expression of transplanted Reporter Genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase Gene (HSV1-tk) as a Reporter Gene and [8- 14 C]-ganciclovir as a Reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8- 14 C]-ganciclovir, As a result, specific accumulation of phosphorylated [8- 14 C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk Gene. Methods: An adenoviral vector was constructed carrying the HSV1-tk Gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8- 3 H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8- 14 C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies. Results: Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a Reporter Gene and [8- 14 C]-ganciclovir as an imaging Reporter probe. A good correlation (r 2 = 0.86) between the [8- 14 C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8- 18 F]-fluoganciclovir and microPET imaging supports further investigation of [8- 18 F]-fluoroganciclovir as a PET Reporter probe for imaging HSV1-tk Gene expression. Conclusion: These results demonstrate the feasibility of using [8- 14 C]-ganciclovir as a Reporter probe for the HSV1-tk Reporter Gene, using an in vivo adenoviral mediated Gene delivery system in a murine model. The results form the foundation for further investigation of [8- 18 F]-fluoroganciclovir for noninvasive and repeated imaging of Gene expression with PET.
Sean W Kennedy - One of the best experts on this subject based on the ideXlab platform.
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a luciferase Reporter Gene assay and aryl hydrocarbon receptor 1 genotype predict the ld50 of polychlorinated biphenyls in avian species
Toxicology and Applied Pharmacology, 2012Co-Authors: Gillian E Manning, Reza Farmahin, Doug Crump, Stephanie P Jones, Jeff Klein, Alex Konstantinov, Dave Potter, Sean W KennedyAbstract:: Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase Reporter Gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 Reporter Gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R(2)≥0.87, p<0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals.
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a luciferase Reporter Gene assay and aryl hydrocarbon receptor 1 genotype predict the ld50 of polychlorinated biphenyls in avian species
Toxicology and Applied Pharmacology, 2012Co-Authors: Gillian E Manning, Reza Farmahin, Doug Crump, Stephanie P Jones, Jeff Klein, Alex Konstantinov, Dave Potter, Sean W KennedyAbstract:Abstract Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase Reporter Gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 Reporter Gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R2 ≥ 0.87, p
