162. Bioluminescence Imaging of a Tetanus Toxin C-Terminal Fusion Protein after Vector Delivery
Authors: Lisa Pike, Sam Wang
Journal: Molecular Therapy (2006)

Firefly luciferase (FLuc) has routinely been used in vitro to quantitate gene expression by measuring the light emitted after theaddition of ATP and luciferin. It is now possible to replace luminometer-based analysis of luciferase activity with digital imaging techniques to measure luciferase activity noninvasively in cells and live animals over time. Previous studies have indicated that a single injection of an HSV amplicon vector (2 μl, 1 × 108 tu/ml) expressing Fluc cDNA under a viral promoter into the mouse cortex was sufficient to detect robust luciferase activity for at least one week. Interestingly, some of the injected animals also appeared to express luciferase activity along the back of the animal, indicating that luciferase expression might be detectable from or around the spinal cord. The ability to modify both pre- and post-synaptic neurons in a pathway could be valuable for potential experimental and therapeutic modalities. We have constructed and characterized an HSV amplicon vector expressing an FLuc-tetanus toxin C-terminal fragment (FLuc::TTC; LT) fusion protein to examine whether this delivery mechanism can be used to assess transynaptic protein spread non-invasively in the CNS. Western blot analysis of infected Gli36 cells confirmed expression of the intact fusion protein and luminometer-based assays confirm luciferase activity. Experiments are ongoing to validate transynaptic transport of Fluc-TTC in both primary culture and brain, and by bioluminescence imaging using various delivery modalities, including intramuscular and intraventricular paradigms. Firefly luciferase (FLuc) has routinely been used in vitro to quantitate gene expression by measuring the light emitted after theaddition of ATP and luciferin. It is now possible to replace luminometer-based analysis of luciferase activity with digital imaging techniques to measure luciferase activity noninvasively in cells and live animals over time. Previous studies have indicated that a single injection of an HSV amplicon vector (2 μl, 1 × 108 tu/ml) expressing Fluc cDNA under a viral promoter into the mouse cortex was sufficient to detect robust luciferase activity for at least one week. Interestingly, some of the injected animals also appeared to express luciferase activity along the back of the animal, indicating that luciferase expression might be detectable from or around the spinal cord. The ability to modify both pre- and post-synaptic neurons in a pathway could be valuable for potential experimental and therapeutic modalities. We have constructed and characterized an HSV amplicon vector expressing an FLuc-tetanus toxin C-terminal fragment (FLuc::TTC; LT) fusion protein to examine whether this delivery mechanism can be used to assess transynaptic protein spread non-invasively in the CNS. Western blot analysis of infected Gli36 cells confirmed expression of the intact fusion protein and luminometer-based assays confirm luciferase activity. Experiments are ongoing to validate transynaptic transport of Fluc-TTC in both primary culture and brain, and by bioluminescence imaging using various delivery modalities, including intramuscular and intraventricular paradigms.