Non-invasive imaging of cell type-specific gene activation using HSV-tk as positron emission tomography reporter gene in combination with the SLC43A3 nucleobase transporter

基因的表达导致蛋白质的生物合成，这些蛋白质完成关键功能，如通过细胞膜运输分子。基因的表达取决于内外因素，并且随着时间的推移而变化。活细胞内特定基因的表达可以借助报告基因进行评估。为此，报告基因被插入到DNA中的一个位置，该位置与要测量其表达的目标基因由相同的启动子控制。然后，目标基因的翻译伴随着报告基因的翻译。在我们的项目中，我们将使用基因报告系统，允许使用正电子发射断层扫描(PET)评估基因表达。报告基因由单纯疱疹病毒1型胸苷激酶(HSV-tk)基因组成。HSV-tk酶使PET示踪分子[18F][18F][FHBG]磷酸化，导致[18F]FHBG在HSV-tk阳性细胞中积聚。因此，可以使用PET非侵入性地定量测量靶基因的表达。用[18F]FHBG在体内监测HSV-tk的表达受到[18F]FHBG无法进入某些细胞类型的限制。[18F]例如，FHBG无法穿过完整的血脑屏障。最近，一个来自日本的研究小组发现了一种名为SLC43A3的基因，它能够选择性地通过细胞膜运输碱基，如[18F]FHBG。基于这些结果，我们假设当HSV-tk与核苷酸转运蛋白SLC43A3结合时，这些限制可以得到解决。为此，我们建立了一个在CFOS启动子控制下表达报告基因HSV-tk的小鼠系，并另外表达了普遍存在的碱基转运蛋白SLC43A3。CFos属于即刻早期基因的一类，是细胞激活的最显著标志。[18F]FHBG应用于清醒的小鼠，在小鼠执行行为任务时积聚。随后的PET测量将显示CFos在活体动物中的任务依赖性表达，从而引入第一个在中枢神经系统工作的PET基因报告系统。此外，通过与DAT-Cre和AgRP-Cre小鼠品系杂交，我们将分别获得在CFos启动子控制下分别在多巴胺能细胞和AgRP细胞中表达HSV-tk的小鼠系。在这些小鼠中，[18F]FHBG的积聚分别在多巴胺能细胞或AgRP细胞中显示出任务依赖的CFos的表达。由此，我们将介绍第一个细胞类型特异性的PET基因报告系统。

Expression of genes leads to the biosynthesis of proteins that fulfil crucial functions, such as transport of molecules through cellular membranes. Gene expression depends on internal and external factors and varies in time. Expression of a specific gene within a living cell can be assessed with the help of reporter genes. For this purpose the reporter gene is inserted into the DNA at a position that is controlled by the same promoter as the target gene, of which the expression is to be measured. Translation of the target gene is then accompanied by translation of the reporter gene. In our project we will use a gene reporter system that allows for the assessment of gene expression with positron emission tomography (PET). The reporter gene consists of the Herpes Simplex Virus-1 Thymidine Kinase (HSV-tk) gene. The enzyme HSV-tk phosphorylates the PET tracer molecule [18F]Fluoro-3-hydroxymethylbutylguanine ([18F][FHBG) and leads to accumulation of [18F]FHBG in HSV-tk positive cells. Thereby, target gene expression can be measured non-invasively and quantitatively using PET. In vivo monitoring of HSV-tk expression with [18F]FHBG is limited by the inability of [18F]FHBG to enter certain cell types. [18F]FHBG, for instance, fails to cross the intact blood brain barrier. Recently, a group from Japan discovered a gene, SLC43A3, that is capable of selectively transporting nucleobases, such as [18F]FHBG, through cellular membranes. Based on these results we hypothesize that the limitations can be solved when HSV-tk is combined with the nucleobase transporter SLC43A3. To this end, we have generated a mouse line that expresses the reporter gene HSV-tk under control of the cfos promoter and additionally expresses ubiquitously the nucleobase transporter SLC43A3. cfos belongs to the class of immediate early genes and is the most prominent marker for cellular activation. [18F]FHBG, applied to the awake mouse, accumulates while the mouse performs a behavioural task. The sub-sequent PET measurement will show task-dependent cfos expression in the living animal and thereby introduce the first PET gene reporter system that works in the central nervous system. Furthermore, by crossing with the DAT-Cre and the AgRP-Cre mouse line, we will generate mouse lines with HSV-tk expression under the control of the cfos promoter exclusively in dopaminergic cells or AgRP cells, respectively. In these mice [18F]FHBG accumulation shows task-dependent cfos expression exclusively in dopaminergic cells or AgRP cells, respectively. Thereby we will introduce the first cell type-specific PET gene reporter system.