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note
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A full-length mouse NMDAR1 cDNA was subcloned into the AAV plasmid from the parent plasmid, pSub201, under the control of a CMV immediate-early promoter and bovine growth hormone (bGH) polyadenylation site between the AAV inverted terminal repeats. Recombinant AAVNMDAR1 virus was generated helper-free as described (11).
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13
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0342414400
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note
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Eight months after vector administration, genomic DNA was extracted from gut, testes, spleen, and liver by standard methods. A 141-base pair (bp) product was amplified with DNA (200 ng), CMV primers (400 nmol), CMV-1 (5′ CCCAGTACATGACCTTATGGG 3′), and CMV-2 (5′ CCAGACTTGGAAATCCCCGT 3′). Analysis of β-actin genomic DNA was used to monitor DNA integrity, using primers β-A1 (5′ CTCTTC-CAGCCTTCCTTCC 3′) and β-A2 (5′ GTCACCTTCAC-CGTTCCAG 3′) to amplify a 772-bp band.
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14
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0342849307
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note
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Serum and antibodies were applied to nitrocellulose membranes for 1 hour at room temperature (RT ) or overnight (O/N) at 4°C following a 90-min incubation in 0.1% Tween 20 containing 5% fetal bovine serum (FBS) in Tris-buffered saline. Antibodies were visualized by incubation for 1 hour at RT with peroxidase-conjugated anti-rat or anti-mouse antibody (1:12,000, Sigma) and ECL detection (Amersham). Hippocampal and cortical extracts were prepared from naïve rat brain. Two preparations were used: (i) a crude hippocampal extract was prepared by homogenization in cold 320 mM sucrose in 10 mM Tris-HCl (pH 7.4); (ii) a nondenatured membrane extract was prepared similarly but in the presence of protease inhibitors (Mini Complete, Boehringer Mannheim). After centrifugation at 7000g for 10 min at 4°C, the resulting supernatant was centrifuged at 37,000g, for 40 min at 4°C and the pellet was resuspended in 10 mM Tris-HCl (pH 7.4) containing protease inhibitors. For serum and corresponding CSF antibody screening, extracts were separated and transferred to polyvinylidene difluoride membrane, and the ECL plus (Amersham) detection system was used.
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15
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0342849308
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note
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Ninety-six-well plates were coated with streptavidin (5 μg/ml at 37°C, O/N) followed by a 2-hour incubation with 1% FBS in 0.1%Tween 20 in PBS (PBST). Each peptide (1.2 nmol reconstituted in 0.2 ml dimethyl sulfoxide; Chiron Technologies, Clayton, Victoria, Australia) was diluted (1:1000 in PBST) before addition to the plates and incubation for 2 hours at RT. AAVNMDAR1, AAVlac, and naïve serum (1:200) were added and incubated at 4°C, O/N. Following a 1-hour incubation with peroxidase-conjugated antirat secondary antibody (1:40,000, RT), OPD substrate (Sigma) was applied and absorption at 490 nm was determined.
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3H]thymidine before harvesting onto filter mats and determination of thymidine incorporation.
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Animals were anesthetized with 60 mg/kg i.p. pentobarbital, and CSF (80 to 100 μl) was drawn from the cisterna magna using a 27-gauge needle. Rats were then left at least 7 days before kainate injection (10 mg/kg i.p) and CSF sampling under anesthesia 2 hours later.
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26
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0342414399
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note
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Naïve (n = 4), AAVlac (n = 4), AAVNMDAR1 (n = 4), and AAVNMDAR1 animals 2 hours after kainate administration (n = 2) were anesthetized before perfusion with 120 ml PBS. Anti-rat IgG immunohistochemistry (1:250, Sigma) was conducted on 16-μm hippocampal sections.
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We thank D. Wu, P. Schweder, J. Francis, S. McPhee, and R. Bland for general technical assistance and advice, C. G. Janson, J. Heywood, M. Dragunow, J. Fraser, and K. Lehnert for scientific assistance, helpful discussions, and manuscript review, T. Hughes for the NMDAR1 cDNA, and X. Xiao and R. J. Samulski for the original AAV cloning and packaging plasmids and advice regarding generation of recombinant AAV vectors. Supported in part by the New Zealand Marsden Fund, New Zealand Health Research Council, and the Jefferson Faculty Foundation.
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