Figure 1

(A) Schematic diagram of the vector constructs pCAG/DT-A, pCAG and pCE-29. Small arrows beneath the pCAG/DT-A plasmid indicate positions and directions of primers used for PCR and RT-PCR. The pCAG/DT-A vector could be identified by PCR using the primer set DTA-S/DTA-2RV. DT-A mRNA synthesized from the CAG promoter can be identified by RT-PCR using the primer set βA-1/DTA-RV. Arrowheads above the CAG promoter indicate the transcription initiation site. Thick line indicates a sequence corresponding to a portion of the 2nd intron, 3rd exon and 3'-noncoding region of rabbit β-globin gene. Red line indicates the sequence of pBluescript SK(-) vector. A portion of the 1st intron of the chicken β-actin gene is shown by a green line. ATG, translation initiation site; CAG, cytomegalovirus enhancer and chicken β-actin gene promoter; DT-A, diphtheria toxin A-chain gene; EGFP, enhanced green fluorescent protein cDNA; pA, polyadenylation site of rabbit β-globin gene; SVpA, polyadenylation sites of SV40 gene. (B) Schematic diagram of the procedure of intravenous injection (IVI) of plasmid DNA/lipid complex and sampling schedule. IVI was performed every day for up to 6 days. Sampling was performed on 4 days (4-d) and 3 (3-w) and 5 weeks (5-w) after the final IVI. 1-d, 1 day. (C) (a,b) EGFP fluorescence in the kidneys from MNCE-36 transgenic mouse (a) and its non-transgenic littermate (b). Glomeruli [indicated by arrows in (a)] and a portion of renal tubules [indicated by arrowheads in (a)] were positive for EGFP fluorescence in the transgenic mouse, but the non-transgenic mouse did not exhibit fluorescence. (c,d) Immunohistochemical staining of the glomeruli from the MNCE-36 mouse (c) and its non-transgenic littermate (d) using anti-GFP antibodies. The surface of the transgenic glomerulus [indicated by arrows in (c)] appeared to have reacted intensely with the antibodies, while the non-transgenic control glomerulus did not react with the antibodies. (e,f) Immunoelectron microscopic analysis of the MNCE-36 kidney reacted with anti-GFP antibodies. Reactive substances (indicated by arrows) were predominantly observed in glomerular epithelium (epi) (e) and slightly in glomerular endothelium (end) (f). b.m., basement membrane; e.l., endothelial lumen.