Figure 3.

Vector characterization of RRV-2A encoding the TKO transgene in U87-MG cells. (A) Replication kinetics of RRV-2A-TKO variants. Viral genomes isolated from the viral supernatant of infected U87-MG cells were quantified by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) using primers spanning the viral envelope region. Infected cells were passaged every other day for 10 days post initial infection, and supernatants were collected at each indicated time point. Data shown represent one of three independent experiments. RRV-IRES-yCD2 vector was included as control. (B) Western blot analysis of viral proteins produced by U87-MG cells maximally infected with RRV-2A-TKO variants. Twenty micrograms of total protein lysates was loaded per well. Membranes were incubated with anti-HSV-1-TK antibody (top panel). Detection of GAPDH using the anti-GAPDH antibody was included as a loading control. Protein standards are indicated in kDa. (C) LD₅₀ of ganciclovir (GCV)-mediated killing of maximally infected U87-MG cells with RRV-2A-TKO variants. Cells were cultured in the presence of GCV in different concentrations for 7 days. Naïve cells were included as a control for GCV cytotoxicity at each concentration. Cell viability was quantified by using MTS assay at 7 days post GCV addition, and the percentage of cell survival was calculated relative to RRV-infected cells not treated with GCV. The data set represents one of the three independent experiments. Error bars indicate the standard deviation of the data set. Numbers in parentheses indicate LD₅₀ values. (D) Stability 2A-TKO transgene cassette from proviral DNA in maximally infected U87-MG cells (15 days post infection). The arrow indicates the size of the PCR product expected for the intact 2A-TKO transgene cassette. The asterisk indicates deletion in the transgene cassette of RRV-T2A-TKO; + controls are PCR product amplified from plasmid DNA corresponding to each RRV-2A-TKO variant. DNA molecular ladder (1 kb plus; Life Technologies) is shown in the first lane of the gel; numbers indicate the size of the ladders in base pair.