Fig. 1.

Generation and characterization of LT-expressing B16/LT tumor cell line. B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian codon-optimized gene encoding the initial 258 amino acids of Merkel cell polyomavirus (strain 350) large T antigen (LT) under the control of a cytolomegalovirus promoter and a GFP reporter under the control of the EF1 promoter to generate tumorigenic B16/LT tumor cell line. (A) Characterization of transduction of B16/LT tumor cells by flow cytometry analysis after sorting. B16/LT tumor cells (Gray line, clear histogram) or control B16 melanoma cells (black line, filled black histogram) were sorted and characterized for GFP expression by flow cytometry analysis. (B) Characterization of LT expression by Western Blot analysis. C57BL/6 mice were vaccinated intramuscularly by electroporation with pcDNA3-LT (10 (μg/20 μl per mouse) three times at a 1-week interval. Two months later, the vaccinated mice were boosted with the same dose and regimen. Western Blot analysis of sera from vaccinated mice was performed 1 week after the last vaccination to characterize LT expression of B16/LT cells. Membranes were probed with either (1) sera from mice vaccinated with pcDNA3-LT or (2) loading control anti-beta actin antibody. Lane A, negative control B16. Lane B, B16/LT. Lane M, size marker.