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The recombinant adenovirus vector AxCA-NT-NFM-myc was constructed by the COS-TPC method (26). Briefly, an expression cosmid cassette (pAxCA-NT-NFMmyc) was constructed by insertion of the expression unit (composed of the cytomegalovirus enhancer plus the chicken β-actin promoter, a complementary DNA coding sequence, and the rabbit β-globin polyadenylation signal sequence) into Swa I site of pAxw, which is a 42-kb cosmid containing a 31-kb adenovirus type 5 genome lacking E1A, E1B, and E3 genes. The expression cosmid cassette and the adenovirus DNA-terminal protein complex (Ad5 dlX DNA-TPC), predigested by Eco T221, were cotransfected into 293 cells [American Type Culture Collection (ATCC); CRL1573] by calcium phosphate precipitation. The recombinant viruses were subsequently propagated with 293 cells, and the viral solution was stored at -80°C. The titers of viral stocks were determined by plaque assay on 293 cells. For in vivo injection experiments, highly concentrated recombinant viral stocks were generated by the method of Kanegae et al. (15).
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11 PFU/ml) recombinant adenovirus vector stock in phosphate buffered saline. Injected animals were killed between 3 and 7 days after surgery. Briefly, they were anesthetized with sodium pentobarbital and fixed by transcardiac perfusion with fresh 4% paraformaldehyde with (for immunoelectron microscopy) or without (for immunocytochemistry) 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). Immediately after perfusion, L4 dorsal roots, gangiia, and sciatic nerves were carefully excised. Consecutive 1-mm segments proceeding from the center of the DRG were removed and placed in the same fixative for an additional 2 hours at room temperature and for a further 12 hours at 4°C. The samples were then transferred to 10% sucrose in the same buffer and incubated for 2 hours at 4°C, after which they were cryoprotected with a graded series of sucrose concentrations, up to 60%, in the same phosphate buffer and then embedded In Tissue-Tek O.C.T. compound (Miles, Elkhart, IN) and frozen. For each segment, serial sections 8 to 10 μm thick were cut on a cryostat and collected. The sections were quenched (in 100 mM glycine) for 30 min at room temperature and then permeabilized and blocked (in 1% Triton X-100,0.1% saponin, and 5% skim milk) for 4 hours at room temperature; both solutions were prepared in 0.1 M phosphate buffer (pH 7.3). Subsequently, sections were incubated overnight at 4°C with a mAb recognizing the c-Myc epitope tag (Myc 1-9E10.2 hybridoma cells; ATCC; CRL1729), followed by a second overnight incubation at 4°C with either fluorescein isothiocyanate-labeled sheep anti-mouse immunoglobulin G (Amersham, Buckinghamshire, UK) for light microscopy, or with FluoroNanogold anti-mouse Fab' fluorescein (Nanoprobes, Stony Brook, NY) tor electron microscopy. All antibodies were diluted in the above-mentioned permeabilizing and blocking solution. Light microscopic analysis was performed by epifluorescent light microscopy (Axiophot; Zeiss, Oberkochen, Germany) or by confocal laser scanning microscopy (MRC-1000); Bio-Rad, Cambridge, MA). For analysis of confocal laser scanning microscope images, we used CoMOS software for image collection and qualitatively oriented image processing operations and MPL software for quantitatively oriented image processing operations. The sections labeled with FluoroNanogold anti-mouse Fab' fluorescein were detected by epifluoresence microscopy, fixed again in 1% glutaraldehyde with 0.2% tannic acid in phosphate buffer, rinsed with distilled water, and subsequently processed for silver enhancement in developer [30% gum arabic, 0.85% hydroquinone, and 0.11% silver nitrate in citrate buffer (pH 7.4)]; see Hayat (27) for detailed procedures. After enhancement, sections were stained with 1% uranyl acetate for 2 hours at room temperature. The samples were dehydrated in a graded series of ethanol concentrations and embedded in Epon 812. Ultrathin sections were cut on a conventional ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (1200EX; JEOL, Tokyo, Japan) at an accelerating voltage of 100 KV.
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We thank I. Saito and Y. Kanegae (Institute of Medical Science, University of Tokyo) and J. Miyazaki (University of Tokyo School of Medicine) for supplying us with their recombinant adenovirus vector system and the CAG promoter, respectively. Their technical advice was essential for the successful construction of our vector. We also thank Y. Komiya and T. Tashiro (Gunma University School of Medicinel for their technical assistance and advice on the microinjection into dorsal root ganglia in vivo; S. Ijuin (Nippon Bio-Rad Laboratories) for his technical assistance in confocal laser scanning microscopy; H. Yorifuji (National Defense Medical College) for his guidance in electron microscopic procedures; J.-P. Julien (Montreal General Hospital Research Institute, Centre for Research in Neuroscience, McGill University) and R. Nixon (McLean Hospital, Harvard Medical School) for helpful comments on the manuscript and useful discussions; Y. Kobayashi (Kyorin University School of Medicine) and T. Funakoshi, S. Kondo, Y. Okada, and other members of the Hirokawa lab for stimulating discussions and invaluable advice throughout; and Y. Kawasaki, H. Sato, H. Fukuda, and Y. Sugaya for their technical and secretarial assistance. Supported by a Special Grant-in-Aid for Scientific Research from the Japan Ministry of Educatton, Science and Culture; a grant from the Institute of Physical and Chemical Research (RIKEN) to N.H.; a Grant-in-Aid for Scientific Research from the Japan Ministry of Education, Science and Culture to S.T.; and grants from the Medical Research Council of Canada and Muscular Dystrophy Association of Canada to A.C.P.
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