Reporter genes have been used by molecular biologists for over a decade to track gene expression in cell culture and in animals. Endogenous gene expression is monitored through the creation of a chimeric fusion gene, in which the promoter of an endogenous gene is coupled to the reporter gene. If the endogenous gene promoter is off, neither the endogenous gene nor the reporter gene are transcribed (because they share the same promoter). Similarly, if the promoter for the endogenous gene is activated, then the reporter gene is transcribed. Through the use of reporter genes such as beta-galactasidase, chloramphenicol acetyl transferase, alkaline phosphatase, and luciferase the study of endogenous genes in vivo has been possible. However, these traditional reporter gene methods are limited by their inability to track the location, magnitude, and persistence of gene expression over time. We are developing quantitative assays to image reporter gene expression using positron emission tomography (PET) in living animals and humans. We propose to study two distinct reporter gene/reporter probe systems. These include (i) the Herpes Simple Type 1 Thymidine Kinase (HSV1-tk) reporter gene/8-[18-F]- fluoroganciclovir (FGCV) reporter probe; (ii) the Tyrosinase (TYR) reporter gene/6-[18-F]Fluoro-L-DOPA (FDOPA) reporter probe. For both these systems we will (a) relate the PET signal to levels of gene expression using both an adenoviral delivery model and a tumor model; (b) develop tracer kinetic models for uptake and trapping of the reporter probe; (c) develop inducible constructs to show induction of gene expression in vivo; (d) construct a dual reporter gene system in which both reporter genes are expressed with the use of an internal ribosomal entry site (IRES). For the HSV1-tk reporter gene we will study mutant genes and alternate reporter probes to enhance the sensitivity of the imaging assay to detect lower levels of gene expression. We will also study transgenic animals which express HSV1-tk. Pre-clinical studies with suicide gene therapy will also be performed. The assays developed through this work should lead to direct methods for the noninvasive study of gene expression in living animals, and allow for the monitoring of gene expression in human gene therapy trials (e.g., trials using suicide gene therapy to destroy brain tumors).
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