Hsieh Chia-Hung, Kuo Jung-Wen, Lee Yi-Jang, Chang Chi-Wei, Gelovani Juri G, Liu Ren-Shyan
Institute of Medical Science, China Medical University, Taichung, Taiwan.
J Nucl Med. 2009 Dec;50(12):2049-57. doi: 10.2967/jnumed.108.061234. Epub 2009 Nov 12.
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.
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.
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.
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.
单纯疱疹病毒1型胸苷激酶（HSV1 - tk）/绿色荧光蛋白（TKGFP）双报告基因以及多模态成像方法在转化分子遗传成像中监测治疗性基因表达、免疫细胞运输和蛋白质 - 蛋白质相互作用方面发挥着关键作用。然而，TKGFP的细胞毒性和低时间分辨率限制了其在需要快速周转报告基因的研究中的应用。本研究的目的是构建一种具有低细胞毒性和高时间分辨率的新型突变TKGFP融合报告基因，用于实时监测体外和体内缺氧及复氧介导的缺氧诱导因子1（HIF - 1）信号转导活性的时间动态和空间异质性。
通过在N端插入核输出信号（NES）序列并在C端融合小鼠鸟氨酸脱羧酶的降解结构域（dMODC）来产生不稳定的TKGFP。通过蛋白质印迹分析、HSV1 - tk酶活性测定和流式细胞术分析来确定TKGFP在活的NG4TL4细胞中的稳定性。通过将NESTKGFP:dMODC连接到由8个缺氧反应元件拷贝组成的启动子上，研究其作为转录报告基因的适用性，该启动子的活性依赖于HIF - 1。由缺氧和复氧介导的动态转录事件通过NESTKGFP:dMODC或TKGFP进行监测，并通过光学成像和PET确定。
与TKGFP不同，NESTKGFP:dMODC在放线菌酮存在下不稳定，并且显示出较短蛋白质半衰期和酶活性。NESTKGFP:dMODC的快速周转以26S蛋白酶体依赖性方式发生。此外，NESTKGFP:dMODC在活细胞中显示出上调表达和低细胞毒性。对缺氧反应性TKGFP和NESTKGFP:dMODC表达的研究表明，作为报告基因，NESTKGFP:dMODC在监测由缺氧和复氧介导的动态转录事件方面比TKGFP具有更好的时间分辨率；就监测目的而言，TKGFP的表达水平并不理想。
在转化分子遗传成像中，NESTKGFP:dMODC作为报告基因，与光学成像和PET一起，能够直接监测转录诱导并易于确定其与其他生化变化的关联。
