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This Article Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Hsieh, C.-H. Articles by Yeh, S.-H. PubMed Articles by Hsieh, C.-H. Articles by Yeh, S.-H.

Short half-life TKGFP fusion reporter gene for use in translational molecular-genetic imaging

¹ China Medical University, Taipei, Taiwan; ² MAGIC/NRPGM, National Yang-Ming University, Taipei, Taiwan; ³ NPCC, Taipei VGH, Taipei, Taiwan

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Objectives: The low temporal resolution and cytototoxicity of reporter protein of HSV1-tk/GFP (TKGFP) limited its use in the translational medicine. This study aims to create a new TKGFP fusion gene with low cytototoxicity and high temporal resolution for real-time monitoring the temporal dynamics and spatial heterogeneity of gene expression events.

Methods: This reporter was developed by adding a nuclear export signal (NES) in the N-terminal end of TKGFP and fusing the degradation domain of mouse ornithine decaroxylase (MODC) to the C-terminal end. This modified TKGFP (NESTKGFP:dMODC), was unstable in the presence of cycloheximide and had a short half-life of protein and enzyme activity determined by western blot analysis, HSV1-TK enzyme activity assay and FACS. The proteasome inhibition assay also confirmed that the rapid turnover of the modified HSV1-TK was processed in a 26S proteasome-dependent manner. The suitability of NESTKGFP:dMODC as a transcription reporter was tested by linking it to a promoter consisting of 8 copies of hypoxia response elements (HRE) whose activity depends on hypoxia -inducible factor-1 (HIF-1).

Results: Cell proliferation assay demonstrated no growth inhibition was observed in NESTKGFP:dMODC-transfected cells. In the hypoxia treatment, both of TK enzyme activity and fluorescent intensity in native TKGFP or NESTKGFP:dMODC correlated with the mRNA expression levels of VEGF and the magnitude of hypoxia stimulations. Re-oxygenation rapidly decreased TK enzyme activity and fluorescence intensity for NESTKGFP:dMODC reporter in vitro and in vivo.

Conclusions: The novel NESTKGFP:dMODC reporter is suitable for the real-time imaging of temporal dynamics and spatial heterogeneity of HIF-1 signal transduction activity in vitro and in vivo.

This Article Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Hsieh, C.-H. Articles by Yeh, S.-H. PubMed Articles by Hsieh, C.-H. Articles by Yeh, S.-H.