Abstract | UNLABELLED: The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/ green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein- protein interactions in translational molecular-genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. METHODS: Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP:dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxia-responsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP:dMODC or TKGFP and determined by optical imaging and PET. RESULTS: Unlike TKGFP, NESTKGFP:dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP:dMODC occurred in a 26S proteasome-dependent manner. Furthermore, NESTKGFP:dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP:dMODC expression showed that NESTKGFP:dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. CONCLUSION: In translational molecular-genetic imaging, NESTKGFP:dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.
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Authors | Chia-Hung Hsieh, Jung-Wen Kuo, Yi-Jang Lee, Chi-Wei Chang, Juri G Gelovani, Ren-Shyan Liu |
Journal | Journal of nuclear medicine : official publication, Society of Nuclear Medicine
(J Nucl Med)
Vol. 50
Issue 12
Pg. 2049-57
(Dec 2009)
ISSN: 1535-5667 [Electronic] United States |
PMID | 19910419
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Hypoxia-Inducible Factor 1
- Recombinant Fusion Proteins
- Green Fluorescent Proteins
- Thymidine Kinase
- Oxygen
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Topics |
- Animals
- Cell Hypoxia
- Cell Line, Tumor
- Enzyme Stability
- Genes, Reporter
(genetics)
- Green Fluorescent Proteins
(genetics, metabolism)
- Herpesvirus 1, Human
(enzymology)
- Humans
- Hypoxia-Inducible Factor 1
(genetics, metabolism)
- Intracellular Space
(metabolism)
- Mice
- Molecular Imaging
(methods)
- Mutation
- Neoplasms
(genetics, metabolism, pathology)
- Oxygen
(metabolism)
- Protein Biosynthesis
- Protein Transport
- Recombinant Fusion Proteins
(genetics, metabolism)
- Signal Transduction
- Thymidine Kinase
(genetics, metabolism)
- Time Factors
- Transcription, Genetic
- Transfection
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