Construction of plasmids for rAAV
and preparation of rAAV
The human a-synuclein gene was cloned into plasmid pAAV-MCS (Stratagene, La Jolla, CA, [pAAV a-synuclein]). Using the protocol provided by the manufacturer, rAAV-a-synuclein and rAAV-enhanced green fluorescent protein (EGFP) were prepared using pAAV-a-synuclein and pAAV-IRES-EGFP (Stratagene) respectively, and the final titres were 4.2 x 1011 genomes/ml (rAAV-a-synuclein) and 5.6 x 1011 genomes/ml (rAAV-EGFP). The number of rAAV genome copies was semi-quantified by PCR within the cytomegalovirus (CMV) promoter region using primers 5'-GACGT CAATAATGACGTATG-3' and 5'-GGTAATAGCGAT GACTAATACG-3'.
Rats (10-week-old) were anesthetized with pentobarbital (40mg/kg body weight, intraperitoneally) and each viral vector suspension was injected into the anterior end of the left SN (3 ml, 5.3 mm posterior and 2.4mm lateral from bregma, 7.4 mm below the dural surface, tooth bar = 4.0 mm) or left lateral cortex (Cx, 2 ml, 1.0mm anterior and 5.0mm lateral from bregma, 3.0mm below the dural surface, tooth bar = 4.0mm). The injection rate was 1 ml/min.
Double immunofluorescence staining was performed by simultaneous incubation of sections with two primary antibodies (4°C for 14 hours), followed by two secondary antibodies (room temperature for 1 hour). The primary antibodies used here were anti-tyrosine hydroxylase (TH, rabbit IgG, 1:1000, Calbiochem), anti-green fluorescent protein (GFP, mouse IgG, 1:100, Sigma), anti-human a-synuclein (clone LB509, 1:500), anti-cleaved caspase-9 (1:500), anti-a-synuclein phos-phorylated at Ser129 (clone Pser129, rabbit IgG, 1:500, Fujiwara et al., 2002) and anti-histone H1 (mouse IgG, 1:500, Santa Cruz Biotechnology).
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