Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis.
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.
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), US National Institutes of Health (NIH), Bethesda, Maryland, USA.
Zhe
Z
Wang
School of Life Science and Technology, Xidian University, Xi'an, China.
Fu
F
Wang
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), US National Institutes of Health (NIH), Bethesda, Maryland, USA.
Naoki
N
Hida
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), US National Institutes of Health (NIH), Bethesda, Maryland, USA.
Dale O
DO
Kiesewetter
School of Life Science and Technology, Xidian University, Xi'an, China.
Jie
J
Tian
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), US National Institutes of Health (NIH), Bethesda, Maryland, USA.
Gang
G
Niu
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), US National Institutes of Health (NIH), Bethesda, Maryland, USA.
Xiaoyuan
X
Chen
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D017930:Genes, Reporter; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D008822:Mice, Transgenic; D010456:Peptides, Cyclic / Q000737:chemistry*; D049268:Positron-Emission Tomography / Q000379:methods*; D015202:Protein Engineering; D013937:Thymidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010456:Peptides, Cyclic; D004317:Doxorubicin; D013937:Thymidine Kinase; D053148:Caspase 3