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For now, ibogaine clinics in Canada are essentially nonexistent. Feeding conditions are as follows: •, ad libitum; ▴, night (NT); ▵, day (DT). Feeding condition is indicated at bottom of each figure. The DNA probes used for identification and characterization of the clone (3′- and 5′-probes) are indicated bythick lines. B, Southern blot analysis of BamHI-digested genomic DNA of PMP22-transgenic and wild-type founder mice using the 5′-probe (lanes 2 and 3 are wild type, andlanes 1, 4, and 5 represent the transgenic lines TgN247, TgN248, and TgN249, respectively). Ten micrometer cross-cryosections of the sciatic nerves of 8-, 21-, and 70-d-old wild-type and PMP22-transgenic mice were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s recommendations. RNA was isolated using the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s recommendations. Digoxigenin-labeled riboprobes were generated from pBluescript SK-vector (Stratagene, La Jolla, CA,) containing the full-length sequence of rMAL cDNA (Schaeren-Wiemers et al., 1995b) with T3 (antisense) and T7 (sense) RNA polymerase, using digoxigenin-UTP (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions. For Buy nembutal online of the transgene copy number, an N-CAM probe (Cremer et al., 1993) was used as internal standard.

After prehybridization of the membrane, PMP22 and P0 mRNA were detected by hybridization with the respective radioactively labeled cDNA probe. Generation of PMP22-transgenic mice.A, Structure of the genomic PMP22 cosmid clone pTCF-6.1. Antibodies directed against PMP22 (Susi 4) (Pareek et al., 1993) or directed against the myelin protein P0 (P0raba; gift from Dr. M. Filbin, Hunter College, NY, NY) were both applied at a dilution of 1:1000 at 4°C for 16 hr. Nerve conduction velocities of control mice were similar to those reported earlier (Low and McLeod, 1975;Pollard and McLeod, 1980; Montag et al., 1994; Adlkofer et al., 1995). Double-sided U test (significance level 0.05) was used to assess the statistical significance of differences in the nerve conduction studies between PMP22-transgenic and control mice (Instat, GraphPad, San Diego, CA). Electrophysiological analysis. iboga for sale for electrophysiological analysis have been described (Adlkofer et al., 1995). Six PMP22-transgenic and five wild-type control animals (sibling of two litters) were examined. Latencies, amplitudes, duration of the compound muscle action potential (CMAP or M-response) (Kimura, 1989), and the F-wave elicitability, latency, and duration were analyzed in the sciatic nerve (Hartung et al., 1988; Kimura, 1989). Because CMAP amplitudes were very low in PMP22-transgenic mice, 10-20 M-responses in these animals were averaged.

Preparation of sciatic nerve homogenates and Western blot analysis. Proliferation analysis. Mice were injected with 5-bromo-2′-deoxyuridine (BrdU) intraperitoneally (50 mg/kg in PBS) at various time points (21 and 70 d after birth). Generation of transgenic mice. Electron microscopy of sciatic nerves of transcardially perfused experimental and control mice (2% PFA and 2% glutaraldehyde in 0.1 mcacodylate buffer) was performed according to Adlkofer et al. For semithin sections, nerves embedded as for electron microscopy were cut in 2 μm sections and stained with alkaline toluidine blue. Tissue preservation and electron microscopy. Animals and tissues. Postnatal and adult Lewis rats were decapitated, and the tissues were rapidly dissected and snap-frozen in liquid nitrogen for RNA extraction or embedded in tissue tek (Sakura, Torrance, CA) and frozen at −40°C in isopentane forin situ hybridization. The tissue was post-fixed in the same fixative overnight, immersed in 30% sucrose, embedded in tissue tek, and frozen at −40°C in isopentane. Zygotes were recovered from the oviduct of superovulated B6C3F1 females mated with males of the same hybrid. ᠎Conte nt was generat ed with　the　he lp of GSA C᠎on tent Gene᠎ra tor  DE MO᠎.

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