Determination of optimal conditions to introduce antigen-encoding mRNA into cells. (A,B) Graded doses of EGFP mRNA were transduced into B16 melanoma cells (H2-K^b) and 5 μg EGFP mRNA to NIH3T3 fibroblasts (H2-K^q). Levels of EGFP expression were evaluated by confocal microscopy (20×/0.7 NA oil objective). (C) A total of 5 μg EGFP mRNA–transduced B16 and NIH3T3 cells were evaluated by flow cytometry for expression of EGFP. The number shown represents the percent of EGFP⁺ cells. Data are representative of 3 independent experiments. (D-F) Whole OVA gene-carrying vector SP64 was linearized, and 5 μg of OVA mRNA was transduced to B16, NIH3T3, or EL4 cells for different incubation periods to determine optimal transduction time (incubation time, 1°). Levels of OVA protein from cell lysates were measured by an ELISA kit (Morinaga Institute of Biological Science) at 2 time points after transduction: 0 hours and 16 hours (incubation time, 2°). Data shown are means plus or minus SEM of 4 mice per group.