Generation of hDEC205 transgenic mice. (A) Schematic of a linearized CD11c promoter-hDEC205 transgene. (B) PCR genotyping strategy for hDEC205 Tg mice, where a set of primers was selected for conserved sequences between mouse and human DEC205. The shorter (165 bp) PCR amplicon is generated from the hDEC205 intronless transgene and the longer (293 bp) from the mouse DEC205 gene. Arrows indicate the positions of F (forward) and R (reverse) primers. (C) A representative gel of 5 hDEC205 Tg founders after PCR of genomic DNA from newborn Tg mouse tails. M is for a lane loaded with molecular weight markers, and bold numbers (17, 42, 43, 44, and 49) indicate the lanes with samples from founder mice carrying the transgene. (D) Localization of hDEC205 in lymph nodes of hDEC205 Tg no. 49 and wild-type mice was visualized by immunohistochemistry. Acetone-fixed cryostat sections of subcutaneous lymph nodes were stained with 3G9 mAb, followed by secondary PE-conjugated antihuman IgG, and FITC-anti-B220 to discriminate B and T areas. Images were taken 200× magnification, but the region with the asterisk (*) is magnified 400× on the right using a Molecular Devices OlympusAX70 deconvolution microscope (Olympus) running METAMORPH Meta Imaging 3.0 (Universal Imaging Corporaration). (E) Expression of hDEC205 in the spleen of wild-type and hDEC205 Tg no. 49 mice. Lymphocyte lineage (CD3, CD19, and DX5) negative cells were surface stained with antibodies for hDEC205 (3G9-Alexa647), CD11c, CD8α, and PDCA1 to monitor hDEC205 expression in PDCs (PDCA1⁺), and cDCs (CD11c⁺ and CD8⁺ or CD8⁻). (F) Skin-draining lymph node cell suspension from wild-type and hDEC205 Tg no. 49 mice were stained with mAbs for hDEC205 (3G9-A647), CD11c, and MHC II to detect hDEC205 in migratory DCs and cDCs. In histograms, blue is for wild-type (WT) and red for hDEC205 Tg mice (Tg; E-F).