Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

¹ Institute of Medical Science, China Medical University, Taichung, Taiwan; Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan
² Institute of Radiological Sciences, Central Taiwan University of Science and Technology, Taichung, Taiwan
³ Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan
⁴ Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan; National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, Taiwan
⁵ Department of Experimental Diagnostic Imaging, University of Texas M.D. Anderson Cancer Center, Houston, Texas
⁶ National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, Taiwan; Department of Nuclear Medicine, Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan

^* To whom correspondence should be addressed. E-mail: rsliu{at}vghtpe.gov.tw.

	Abstract

Herpes simplex virus type 1 thymidine kinase (HSV1-TK) is a widely used reporter for in vivo noninvasive monitoring of therapeutic gene expression, immune cell trafficking, and protein–protein interactions in various animal systems. However, the stability of HSV1-TK limits its application in studies that require rapid turnover of the reporter. The purpose of this study was to create a destabilized HSV1-TK as a transcription reporter that allows for dynamic studies of short-time-scale gene expression events. Methods: A destabilized HSV1-TK was created by targeting inactivating mutations in the nuclear localization signal of HSV1-TK and fusing the degradation domain of mouse ornithine decarboxylase to the C-terminal end. The protein or enzyme stability was determined by Western blot analysis and HSV1-TK enzyme activity assay, respectively. The proteasome inhibition assay was used to test whether the rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome–dependent manner. The suitability of destabilized HSV1-TK as a transcription reporter was tested by linking it to a tetracycline-turnoff–expressing system. The dynamic transcriptional events mediating a series of doxycycline inductions were monitored by destabilized HSV1-TK or by native HSV1-TK and were determined by an in vitro HSV1-TK enzyme activity assay and in vivo small-animal PET imaging. Results: The destabilized HSV1-TK, unlike wild-type HSV1-TK, was unstable in the presence of cycloheximide and had a short half-life of protein and enzyme activity. The rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome–dependent manner. Furthermore, the destabilized HSV1-TK had low cytotoxicity when it was highly expressed in living cells. The results of dynamic gene expression studies in vitro and in vivo showed that the destabilized HSV1-TK is an optimal reporter for monitoring short-time-scale dynamic transcriptional events mediating a series of doxycycline inductions, whereas the wild-type HSV1-TK is not optimal to achieve this purpose. Conclusion: The use of destabilized HSV1-TK as a transcription reporter together with a molecular probe, which has a short physical and biologic half-life, allows more direct monitoring of transcription induction and easier monitoring of its coincidence with other biochemical changes.

Key Words: herpes simplex virus type 1 thymidine kinase (HSV1-TK), mouse ornithine decarboxylase (MODC), doxycycline (Dox), nuclear localization signal (NLS), PET imaging

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