Yaghoubi Shahriar S, Gambhir Sanjiv S
Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Clark Center, 318 Campus Drive, E150, Stanford, CA 94305-5427, USA.
Nat Protoc. 2006;1(4):2137-42. doi: 10.1038/nprot.2006.334.
The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.
单纯疱疹病毒1型胸苷激酶（HSV1-tk）正电子发射断层扫描（PET）报告基因（PRG）或其突变体HSV1-sr39tk用于研究培养细胞中的细胞内分子事件，以及在活体中对细胞内分子事件和细胞运输进行成像。有两种体外方法可用于检测细胞或组织中HSV1-tk或HSV1-sr39tk的基因表达。一种方法是在固定的孵育时间后，通过测量在固定量的细胞裂解物蛋白中被这些酶磷酸化的放射性标记底物的量，来确定细胞或组织裂解物中HSV1-TK或HSV1-sr39TK酶的活性水平。另一种方法称为“细胞摄取测定法”，除了HSV1-TK或HSV1-sr39TK活性水平外，还考虑了特定细胞对放射性标记底物的天然摄取和流出特性。在活体研究对象中进行研究之前，这两种测定法均可用于验证培养细胞中的分子模型。每种测定法均可在一天内完成。
