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

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.

Noninvasive bioluminescence imaging of the dynamics of sanguinarine induced apoptosis via activation of reactive oxygen species

“…It is noteworthy that although BLI have proved effective in preclinical trials, it is yet to be fully validated as useful tool in clinical applications. Radionuclide reporter probes such as cTK are more promising for visualizing apoptosis and drug response in a clinical setting [ 32 , 33 ]. Our future work will proceed towards the clinical translation of the cTK probe for apoptosis-related drug development and therapeutic evaluation.…”

“…However, due to the low sensitivity, some of these probes are not well suitable for real-time visualization of caspase-3 activation and screening the candidate drugs for dysregulated apoptosis in vivo . Recently Kanno A et al and our group have developed two novel apoptosis reporter probes, cyclic firefly luciferase (cFluc) [ 30 , 31 ] and cyclic HSV1-TK (cTK) [ 32 , 33 ], by fusing two fragments of DnaE inteins to neighboring ends of luciferase or TK connected with DEVD. Upon cells undergoing apoptosis stimulated by exogenous drugs, caspase-3 is activated and cleaves the genetically encoded cFluc or cTK, leading to the restoration of luciferase or thymidine kinase activity that can be detected in living organisms by bioluminescence imaging (BLI) or positron emission tomography (PET) imaging.…”

Nanomaterials in Neural‐Stem‐Cell‐Mediated Regenerative Medicine: Imaging and Treatment of Neurological Diseases

“…The second class is an enzyme‐based system, using reporter genes to produce specific enzymes that modify the radiolabel by inhibiting its release from a cell. The herpes simplex virus thymidine kinase type 1 (HSV1‐tk) is the most common reporter gene for radionuclide imaging . The third class is a type of reporter gene, which is encoded by radiolabel transport proteins.…”