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. Author manuscript; available in PMC: 2022 Aug 24.
Published in final edited form as: Cancer Gene Ther. 2022 Feb 10;29(8-9):1240–1251. doi: 10.1038/s41417-022-00431-y

Figure 4. Therapeutic transgenes in the oFV backbone are stable for up to 3 in vitro passages, TK cDNA appears to be more stable than iCasp9 cDNA in the oFV backbone during in vivo amplification:

Figure 4

A. oFV-TK and oFV-iCasp9 were serially passaged five times at a fixed MOI=0.5 in U251-U3-mCherry-U3-luc cells and at the end of each passage (day 8 post infection) the genomic DNA was isolated from the infected cells for a PCR amplification of the bel2 region (containing the transgene) or env (control). B., D. U251-U3-mCherry-U3-luc tumors from mice infected with oFV-TK/oFV-iCasp9 and treated with GCV (n=3)/AP20187 (n=2) or PBS control (n=3 for oFV-TK and n=2 for oFV-iCasp9) were explanted and cultured in vitro, then genomic DNA was isolated from the explants and PCR analyzed for the presence of the oFV-TK (B) or oFV-iCasp9 (D) provirus (env) as well as the intact transgene cDNA (bel2 region), C. qPCR reaction was performed on the DNA isolated from oFV-TK infected tumors to determine the env and TK copy number, result is normalized to endogenous β-actin copy number. +c – positive control (oFV-TK or –iCasp9 infectious clone DNA), −C – negative control (genomic DNA from uninfected U251-U3-mCherry-luc cells), NTC- no template control, P1-P5 – passage number, i-iv – specific tumors collected from the treated mice for analysis.