Future Directions

Project Completion

Continued work will finish assembling the viral vector design via homologous recombination in E. Coli MW001 strain. PCR and sequencing the resulting plasmid will confirm the BAC identity and the genetic integrity.

Characterization

Validation of the TN7 integration with GFPuv as a gene expression reporter will demonstrate the proof of concept. The plasmid will be transfected into an expression cell line to produce the HSV recombinant virus. The resulting virus will be used to infect Vero-E6 cells to observe delivery and expression. GFP expression intensity will be quantified using fluorescence microscopy. Cytotoxicity will be determined using a viral titer and in vitro assays. The length of expression will also be observed. If required, improvements in expression will be fine tuned with different promoters and expression cell lines.

Application : Neural Tracing

After validation in vitro , the recombinant virus will be injected into the central nervous system of in vivo mouse models with replication ability to demonstrate use as a neural tracer. Genetic modification of the recombinant virus will be expanded to include Mcherry and DSred to label subcellular organelles. Based on the protocol and expression time observed in vero cells, the central nervous systems of the mice will be harvested and sliced into cross-sections for fluorescent imaging.

Additional projects could be performed with multigene pathway integration to demonstrate a therapeutic effect. Effectiveness of inserting these therapeutic pathways will be determined with in vitro cellular assays, and will be modulated in future projects with pathway engineering techniques.