Gene gun transfection induces high transgene expression by DCs compared with other naked DNA transfection methods. (A) Luciferase expression by DCs after transfection with naked DNA using lipofection (Lipof), Ca₂PO₄, electroporation (Elec), or gene gun (GG), or after infection with rAd-Luciferase (rAd-Luc). Although rAd-Luc induces the highest luciferase expression, highly significant expression is found with the gene gun compared with other nonviral methods used. Means ± 1 SD from 3 independent experiments are shown. *indicates P < .01. (B) Expression of cytoplasmic OVA by DCs transfected with different naked DNA methods or infected with rAd. Numbers in graphs are percentages of OVA-positive DCs and mean fluorescence intensity (MFI) (in parentheses). (C-D, insets) High transgene expression (OVA-TR⁺ DCs) correlated with the presence of gold particles (arrows) free in the cytosol of DCs (shot directly) (C). Transgene-negative DCs (OVA-TR^– DCs) exhibited fewer gold particles inside membrane vesicles (likely acquired from the medium by endocytosis) (D). Panels C and D, TEM (original magnification × 3000; insets, original magnification × 14 000).