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. 2018 Apr 1;29(4):437–451. doi: 10.1089/hum.2017.205

Figure 6.

Figure 6.

Vector characterization of RRVs expressing multiple transgenes from 2A peptides in U87-MG cells. (A) Schematic diagram of the RRV constructs encoding multiple-transgene cassette of gT2A-hGMCSF-gP2A-yCD2 and gT2A-yCD2-gP2A-hGMCSF. The approximate size of the multiple transgene cassette is shown in parentheses. (B) Western blot analysis of yCD2 proteins produced by U87-MG cells maximally infected with RRV-gT2A-hGMCSF-gP2A-yCD2 and RRV-gT2A-yCD2-gP2A-hGMCSF. RRV-IRES-yCD2 and RRV-IRES-hGMCSF were included as controls. Twenty micrograms of total protein lysates was loaded per well. Membranes were incubated with anti-yCD2 antibody. Detection of GAPDH using the anti-GAPDH antibody was included as a loading control. (C) LD50 of 5-FC-mediated killing of maximally infected U87-MG cells with RRV-2A-yCD2 variants. Cells were cultured in the presence of 5-FC in different concentrations for 7 days. Naïve cells were included as a control for 5-FC cytotoxicity at each concentration. Cell viability was quantified by using MTS assay at 7 days post 5-FC addition, and the percentage of cell survival was calculated relative to RRV-infected cells not treated with 5-FC. 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. Statistical significance was determined by two-way ANOVA. (D) hGMCSF in the supernatant produced from maximally infected U87-MG cells was quantified by ELISA. Error bars indicate the standard deviation of the data set. (E) Stability of yCD2-hGMCSF and hGMCSF-yCD2 transgene cassettes from proviral DNA in maximally infected U87-MG cells (16 days post infection). The arrow indicates the size of the PCR product expected for the intact yCD2-hGMCSF and hGMCSF-yCD2 transgene cassette + controls are PCR product amplified from plasmid DNA corresponding to each RRV-2A-TKO variants. 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 pairs.