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Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis

Nature Protocols volume 10, pages 807–821 (2015)Cite this article

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Abstract

Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we describe the design and use of an engineered cyclic herpes simplex virus 1–thymidine kinase (HSV1-TK) PET reporter whose kinase activity is specifically switched on by apoptosis. The expression of cyclic TK (cTK) in healthy cells leads to inactive product, whereas the activation of apoptosis through the caspase-3 pathway cleaves cTK, thus restoring its activity and enabling PET imaging. In addition to detailing the design and construction of the cTK plasmid in this protocol, we include assays for evaluating the function and specificity of the cTK reporter in apoptotic cells, such as assays for measuring the cell uptake of PET tracer in apoptotic cells, correlating doxorubicin (Dox)-induced cell apoptosis to cTK function recovery, and in vivo PET imaging of cancer cell apoptosis, and we also include corresponding data acquisition methods. The time to build the entire cTK reporter is ∼2–3 weeks. The selection of a stable cancer cell line takes ∼4–6 weeks. The time to implement assays regarding cTK function in apoptotic cells and the in vivo imaging varies depending on the experiment. The cyclization strategy described in this protocol can also be adapted to create other reporter systems for broad biomedical applications.

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Figure 1: cTK reporter system design for apoptosis PET imaging.
Figure 2: 18F-FHBG uptake assay from different cTK variants.
Figure 3: Transfection of cTK266-containing cells with firefly luciferase (fLuc) as the cell proliferation indicator.
Figure 4: Characterization of cTK266 reporter in 22B cells.
Figure 5: Caspase-3 specificity evaluation of cTK266 in cells.
Figure 6: In vivo PET imaging and BLI of apoptotic cells after Dox treatment.

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Acknowledgements

This work was supported, in part, by the Center for Neuroscience and Regeneration Medicine Program at the Henry M. Jackson Foundation, and by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH). We thank T. Ozawa (University of Tokyo) for providing us with the cDNAs of DnaE and PEST.

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Author notes

  1. Zhe Wang and Fu Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), US National Institutes of Health (NIH), Bethesda, Maryland, USA

    Zhe Wang, Naoki Hida, Dale O Kiesewetter, Gang Niu & Xiaoyuan Chen

  2. School of Life Science and Technology, Xidian University, Xi'an, China

    Fu Wang & Jie Tian

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  1. Zhe Wang
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Contributions

Z.W., N.H., G.N. and X.C. conceived and designed this research; Z.W., F.W., N.H. and G.N. performed the experiment; D.O.K. contributed new reagents and analytical tools; Z.W., F.W., G.N., J.T. and X.C. analyzed the data; and Z.W., G.N. and X.C. wrote the manuscript.

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Correspondence to Gang Niu or Xiaoyuan Chen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Real-time PCR analysis cTK variant expression level in different stable selected cell colonies.

The number under each bar indicates the cTK variant at specific split sites, and the number in the bracket identifies the cell colony (n=3).

Supplementary Figure 2 Illustration of the stable cell selection procedure.

(Not drawn to scale.)

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Supplementary Figures 1 and 2 (PDF 238 kb)

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Wang, Z., Wang, F., Hida, N. et al. Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis. Nat Protoc 10, 807–821 (2015). https://doi.org/10.1038/nprot.2015.048

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