164. Bioluminescence Imaging in the CNS after HSV Amplicon Vector Delivery

Abstract

Firefly luciferase has routinely been used to quantitate and analyze gene expression in vitro by measuring the light emitted from a test sample after the addition of ATP and luciferin. It is now possible to extend luminometer-based analysis of luciferase activity by measuring luciferase activity directly in live animals over time using digital imaging techniques. Detection of luciferase activity in the central nervous system (CNS) is possible after vector delivery and substrate administration. A single injection into the mouse cortex of an HSV amplicon vector (2 l, 1 108 tu/ml) expressing firefly luciferase cDNA (FLuc) from either a viral (CMV) or hybrid promoter (CBA) was sufficient to generate robust luciferase activity for at least one week. Luciferase activity was also observed in the mouse cortex using a novel HSV amplicon vector, which expresses FLuc under a regulatable promoter, for up to 5 days after a single induction dose. In the absence of induction, no luciferase activity was observed. Interestingly, some of the injected animals appeared to express luciferase activity along the back of the animal indicating that, perhaps, luciferase gene expression might be detectable from or around the spinal cord. We are currently analyzing these tissues for luciferase expression by immunohistochemistry. Experiments are also underway to determine the source of this signal. By combining bioluminescence and gene delivery techniques we can quickly evaluate luciferase expression in the spinal cord. HSV amplicon vectors will be injected into the lateral ventricle (delivery via the CSF) and into several muscle regions (delivery via retrograde transport of the vector to lower motor neruon cell bodies) for bioluminescence imaging and histology.