Generation of destabilized herpes simplex virus type 1 thymidine kinase as transcription reporter for PET reporter systems in molecular genetic imaging.
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.


CONCLUSIONS
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.
Hsieh
Chia-Hung
CH
Institute of Medical Science, China Medical University, Taichung, Taiwan.
Chen
Fu-Du
FD
Wang
Hsin-Ell
HE
Hwang
Jeng-Jong
JJ
Chang
Chi-Wei
CW
Lee
Yi-Jang
YJ
Gelovani
Juri G
JG
Liu
Ren-Shyan
RS
D000818:Animals; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D004795:Enzyme Stability; D005260:Female; D017930:Genes, Reporter; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D051379:Mice; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism; D049268:Positron-Emission Tomography; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D023041:Xenograft Model Antitumor Assays
D019913:Nuclear Localization Signals; D003513:Cycloheximide; D013937:Thymidine Kinase; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D009955:Ornithine Decarboxylase; D004318:Doxycycline