In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

Hsieh, Chi-Hsun; Liu, Ru‐Shi; Wang, Hsin Ell; Hwang, Jeng Jong; Deng, Win Ping; Chen, Jyh Cheng; Chen, Fu Du

doi:10.1016/j.nucmedbio.2006.04.001

Cited by 10 publications

(12 citation statements)

References 25 publications

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“…The HSV1-TK enzyme activity assay was based on a protocol described elsewhere (5). Briefly, the stored cells were lysed in a lysis buffer and then the cell extracts were incubated for 60 min with 8-3 H-ganciclovir to determine the formation of phosphorylated 8-3 H-ganciclovir.…”

Section: Hsv1-tk Enzyme Activity Assaymentioning

confidence: 99%

“…The procedures for establishing a tetracycline-turnoff-expressing cell line are detailed in our previous report (5). Briefly, the NG4TL4 cells infected with pRevtet-off virus were separately subjected to the infectious supernatant containing pRevTRE-mNLSHSV1-TK: dMODC virus, pRevTRE-mNLSHSV1-TK, and pRevTRE-HSV1-TK virus, and then the cells were selected with 600 mg of hygromycin per milliliter to establish a tetracycline-turnoff-mNLSHSV1-TK: dMODC-expressing cell line (NG4TL4/tet-off-mNLSHSV1-TK: dMODC), a tetracycline-turnoff-mNLSHSV1-TK-expressing cell line (NG4TL4/tet-off-mNLSHSV1-TK), and a tetracycline-turnoff HSV1-TK-expressing cell line (NG4TL4/tet-off-HSV1-TK), respectively.…”

Section: Tetracycline-turnoff-expressing Mnlshsv1-tk: Dmodc and Mnlshmentioning

confidence: 99%

“…This reporter system has been widely used in noninvasive monitoring of therapeutic gene expression in vivo and in immune cell trafficking and protein-protein interactions in various living animals (2-4). However, our previous study showed that HSV1-TK is highly stable in mammalian cells (5). This stability limits its application for monitoring short-time-scale dynamic processes, such as kinetic gene expression controlled by inducible promoters or by a less stable protein with a more rapid turnover.…”

mentioning

confidence: 99%

“…This stability limits its application for monitoring short-time-scale dynamic processes, such as kinetic gene expression controlled by inducible promoters or by a less stable protein with a more rapid turnover. Thus, the highly stable reporter protein of HSV1-TK poses a significant disadvantage for dynamic studies of short-time-scale gene expression events because of poor temporal resolution (5). Furthermore, the HSV1-TK enzyme has been shown to have cytotoxicity on its own when used as a selection marker or therapeutic gene in transgenic mice or in gene therapy strategies because of the nuclear tropism of its protein and the presence of a putative cryptic testis-specific promoter (6)(7)(8).…”

mentioning

confidence: 99%

“…In the bioinformatics analysis of the HSV1-TK amino sequence, there are no potential proteolytic cleavage sites and the protein half-life of HSV1-TK was estimated to be about 30 h on the basis of the N-end rule. This may explain in part why HSV1-TK is stable in mammalian cells (5).…”

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confidence: 99%

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Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

Hsieh¹,

Chen²,

Wang³

et al. 2007

J Nucl Med

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Herpes simplex virus type 1 thymidine kinase (HSV1-TK) is a widely used reporter for in vivo noninvasive monitoring of therapeutic gene expression, immune cell trafficking, and proteinprotein interactions in various animal systems. However, the stability of HSV1-TK limits its application in studies that require rapid turnover of the reporter. The purpose of this study was to create a destabilized HSV1-TK as a transcription reporter that allows for dynamic studies of short-time-scale gene expression events. Methods: A destabilized HSV1-TK was created by targeting inactivating mutations in the nuclear localization signal of HSV1-TK and fusing the degradation domain of mouse ornithine decarboxylase to the C-terminal end. The protein or enzyme stability was determined by Western blot analysis and HSV1-TK enzyme activity assay, respectively. The proteasome inhibition assay was used to test whether the rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasomedependent manner. The suitability of destabilized HSV1-TK as a transcription reporter was tested by linking it to a tetracyclineturnoff-expressing system. The dynamic transcriptional events mediating a series of doxycycline inductions were monitored by destabilized HSV1-TK or by native HSV1-TK and were determined by an in vitro HSV1-TK enzyme activity assay and in vivo small-animal PET imaging. Results: The destabilized HSV1-TK, unlike wild-type HSV1-TK, was unstable in the presence of cycloheximide and had a short half-life of protein and enzyme activity. The rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome-dependent manner. Furthermore, the destabilized HSV1-TK had low cytotoxicity when it was highly expressed in living cells. The results of dynamic gene expression studies in vitro and in vivo showed that the destabilized HSV1-TK is an optimal reporter for monitoring short-time-scale dynamic transcriptional events mediating a series of doxycycline inductions, whereas the wild-type HSV1-TK is not optimal to achieve this purpose. Conclusion: The use of destabilized HSV1-TK as a transcription reporter together with a molecular probe, which has a short physical and biologic half-life, allows more direct monitoring of transcription induction and easier monitoring of its coincidence with other biochemical changes.

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Section: Hsv1-tk Enzyme Activity Assaymentioning

confidence: 99%

Section: Tetracycline-turnoff-expressing Mnlshsv1-tk: Dmodc and Mnlshmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

Hsieh¹,

Chen²,

Wang³

et al. 2007

J Nucl Med

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Radiosynthesis andin vitroevaluation of 1‐(tetrahydro‐5‐hydroxy‐6‐(hydroxymethyl)‐2H‐pyran‐3‐yl)‐5‐[¹²⁵I]iodouracil: A new potential agent for HSV1‐tk reporter gene monitoring

Kim

El‐Gamal

et al. 2010

Labelled Comp Radiopharmac

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a,bÃSynthesis, radiolabelling, and in vitro evaluation of a new 125 I-labelled iodouracil hexitol nucleoside analogue are reported. The target compound was successfully synthesized by an iodination-destannylation method and then purified by reverse phase HPLC. The radiochemical purity of the product was 499% with decay-corrected yields of 4873%. In vitro cellular uptake testing was carried out using MCA and MCA-tk cell lines for comparison of compound 1 with [18 F]FHBG. The newly synthesized compound 1 showed higher accumulation in herpex simplex virus type 1 thymidine kinase (HSV1-tk) gene expression cell line (MCA-tk cell line) than in the wild type MCA cell line compared with [18 F]FHBG. The MCA-tk to MCA cellular uptake ratio for compound 1 was higher than that of [ 18 F]FHBG from 2 h after incubation. The radioiodine-labelled compound 1 (I-125, t 1/2 = 59.37 days) has a longer physical half-life than F-18-(t 1/2 = 110 min) labelled FHBG. Radioiodinelabelled compound 1 could be used for monitoring gene expression for a long time. The selectivity for MCA-tk cell line makes compound 1 a promising imaging agent for HSV1-tk expression.

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Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application

Ottobrini

Ciana

Moresco

et al. 2007

Eur J Nucl Med Mol Imaging

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In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

Cited by 10 publications

References 25 publications

Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

Radiosynthesis andin vitroevaluation of 1‐(tetrahydro‐5‐hydroxy‐6‐(hydroxymethyl)‐2H‐pyran‐3‐yl)‐5‐[¹²⁵I]iodouracil: A new potential agent for HSV1‐tk reporter gene monitoring

Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application

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In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

Cited by 10 publications

References 25 publications

Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

Radiosynthesis andin vitroevaluation of 1‐(tetrahydro‐5‐hydroxy‐6‐(hydroxymethyl)‐2H‐pyran‐3‐yl)‐5‐[125I]iodouracil: A new potential agent for HSV1‐tk reporter gene monitoring

Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application

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Radiosynthesis andin vitroevaluation of 1‐(tetrahydro‐5‐hydroxy‐6‐(hydroxymethyl)‐2H‐pyran‐3‐yl)‐5‐[¹²⁵I]iodouracil: A new potential agent for HSV1‐tk reporter gene monitoring