Tissue distribution of lentivector obtained after in vivo administration into footpads of mice. (a) Detection of integrated lentivector at the site of injection (foot), the draining lymph node, and the spleen but not in the mandibular lymph node and the tip of the tail. Vector integration was detected by amplification of vector-derived GFP sequence with specific primers (arrows) using seminested PCR. dLN, draining lymph node; spl, spleen; man, mandibular lymph node. (b) Histological analysis of GFP expression after administration of lentivector into one hind footpad. The upper panel shows GFP fluorescence of frozen sections of popliteal lymph nodes 2.5 days after injection of CMV-GFP lentivector (lvGFP) or PBS (nontreated) into the footpad. The contralateral popliteal lymph node (contra) of a treated mouse is also shown. Magnification, ×100. The lower panel shows double immunofluorescence analysis of draining lymph nodes and spleen 2.5 days after injection of CMV-GFP lentivector using anti-GFP antibodies (green) counterstained with anti-CD11c, anti-CD11b, and anti-B220 antibodies (red). Magnification, ×400. The use of a CMV-GFP lentivector for immunofluorescence was necessary, because with the bicistronic vaccine constructs (antigen-IRES-GFP), GFP fluorescence was too low to be picked up in histology. For PCR analysis of the GFP sequence, the actual vaccine vector was used.