Generation of ES-DCs expressing OVA antigen and in vivo priming of OVA-specific cytotoxic lymphocytes.
(A) Cre/mutant lox–mediated targeted integration of the expression cassette to a gene-trapped locus is schematically depicted. The expression cassette, the OVA cDNA driven by the chicken β-actin promoter (pCA-OVA) ligated with the puromycinN-acetyltransferase gene driven by the pgk promoter (pgk-Puro-R), was inserted to the gene-trapped site, replacing the β-geo sequence by the effect of Cre recombinase transiently expressed from pCAGGS-Cre. The combination of a lox66 of the replacement vector and a lox71 of a trapped locus resulted in a double-mutant lox, which inhibited further recombination of the replaced allele and increased the frequency of proper recombinant ES cell clones. (B) Stimulation of OVA-specific T-cell hybridoma, RF33.70, with ES-DCs with (○) or without (■) expression of OVA was analyzed. As a control, bone marrow–derived DCs pulsed with 10 μM (⋄) or 1 μM (▪) of OVA257-264 for 4 hours or left unpulsed (▵) are also used as stimulators. Stimulators and hybridomas were cocultured for 24 hours, and IL-2 produced by 2B4 was measured by proliferation of CTLL-20 cells. Results were expressed as mean cpm of triplicate cultures ± standard deviation. (C) ES-DCs with (ES-DC-OVA) or without (ES-DC) expression of OVA protein were injected intraperitoneally on days 0 and 7 into syngenic F1 mice. Splenocytes from the injected mice were harvested on day 14 and cultured in the presence of OVA257-264 (0.1 μM) for 5 days. The resultant cells were assayed for the capacity to lyse EL-4 tumor cells either pulsed with 10 μM OVA peptide (▪) or left unpulsed (■). Results were expressed as mean specific lysis of triplicate assays and the standard deviation of triplicates was less than 2.5%.
