Figure 5. oFV-GFP extensively propagates in subcutaneous indicator glioblastoma tumors in vivo and facilitates long term intratumoral transgene expression without signs of significant transgene loss:

5 million U251-U3-mCherry-U3-luc cells were implanted subcutaneously in CB17-SCID mice and infected intratumorally with PBS control or 4 doses of 5*10⁵ IU oFV-GFP, A. Average oFV-GFP-induced radiance in the oFV-GFP-infected tumors, B. Immunofluorescence staining for mCherry and GFP in sections of PBS-treated and oFV-GFP infected tumors at different time points post infection (mCherry expression is a marker of oFV-infected indicator tumor cells, GFP is a marker of oFV-GFP infection), C. Relative abundance of oFV-GFP transcripts in control and oFV-GFP-infected tumors determined with RNAseq. D. Total DNA was isolated from oFV-GFP infected tumors harvested 39 (n=2) and 66 (n=2) days post infection as well as a PBS-treated tumor. The DNA was PCR analyzed for the presence of proviral (env) and GFP transgene (bel2) DNA. +C – positive, oFV-GFP infectious clone was used as template for the PCR reaction, 39 and 66 dpi – DNA from oFV-GFP infected tumors harvested 39 and 66 days post infection, PBS- DNA from a PBS treated tumor. E. qPCR reaction was performed on the DNA isolated from the oFV-GFP infected and PBS treated tumors as well as U251-U3-mCherry-U3-luc cells infected in vitro at MOI=1 (control) to determine the env and GFP copy number, result is normalized to endogenous β-actin copy number.