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

基因的表达导致完成关键功能的蛋白质的生物合成，例如分子通过细胞膜的运输。基因表达取决于内部和外部因素，并随时间变化。活细胞内特定基因的表达可以在报告基因的帮助下进行评估。为此目的，将报告基因插入DNA中受与靶基因相同的启动子控制的位置，其中靶基因的表达将被测量。然后，靶基因的翻译伴随着报告基因的翻译。在我们的项目中，我们将使用一个基因报告系统，允许评估基因表达与正电子发射断层扫描（PET）。报告基因由单纯疱疹病毒-1胸苷激酶（HSV-tk）基因组成。酶HSV-tk磷酸化PET示踪分子[18 F]氟-3-羟甲基丁基鸟嘌呤（[18 F][FHBG），并导致[18 F]FHBG在HSV-tk阳性细胞中蓄积。因此，可以使用PET非侵入性地和定量地测量靶基因表达。 用[18 F]FHBG体内监测HSV-tk表达受到[18 F]FHBG不能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，日本的一个研究小组发现了一个基因SLC 43 A3，它能够选择性地通过细胞膜转运核碱基，如[18 F]FHBG。基于这些结果，我们假设当HSV-tk与核碱基转运蛋白SLC 43 A3组合时，可以解决这些限制。为此，我们已经产生了一种小鼠系，其在cfos启动子的控制下表达报告基因HSV-tk，并且还普遍表达核碱基转运蛋白SLC 43 A3。cfos属于立即早期基因类别，是细胞激活的最重要标志物。[18F]FHBG，应用于清醒的小鼠，在小鼠执行行为任务时积累。亚PET测量将显示活体动物中的任务依赖性cfos表达，从而引入第一个在中枢神经系统中工作的PET基因报告系统。此外，通过与DAT-Cre和AgRP-Cre小鼠系杂交，我们将产生分别在多巴胺能细胞或AgRP细胞中具有在cfos启动子控制下的HSV-tk表达的小鼠系。在这些小鼠中，[18 F]FHBG蓄积显示任务依赖性cfos表达分别仅在多巴胺能细胞或AgRP细胞中表达。因此，我们将介绍第一个细胞类型特异性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.