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Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro

Nature Protocols volume 1, pages 2137–2142 (2006)Cite this article

Abstract

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.

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Figure 1
Figure 2: Correlation between TK activity in C6 cells and levels of Ad-CMV-HSV1-sr39tk infecting C6 cells.
Figure 3: Cell-uptake studies.

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Acknowledgements

We would like to acknowledge the contributions of Meera Iyer, Eileen Bauer and Khoi Nguyen over the past decade to developing protocols for the detection of PET reporter genes in our laboratory. We also acknowledge the following funding sources: NIH grants: NCI ICMIC P50 CA114747, SAIRP R24 CA92865, P50 CA86306, R01 CA82214; DOE contract DE-FC03-87ER60615; RSNA Postdoctoral Fellowship in Basic Radiological Sciences.

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  1. Departments of Radiology & Bioengineering, Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Clark Center, 318 Campus Drive, E150, Stanford, 94305-5427, CA, USA

    Shahriar S Yaghoubi & Sanjiv S Gambhir

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Correspondence to Sanjiv S Gambhir.

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Yaghoubi, S., Gambhir, S. Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro. Nat Protoc 1, 2137–2142 (2006). https://doi.org/10.1038/nprot.2006.334

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