Generation of Cre- loxP Conditional α-Synuclein Transgenic Mice. (A) Gene targeting strategy for generating conditional ROSA26-αSyn transgenic mice. Human α-synuclein cDNAs were placed downstream of a loxP-flanked cassette containing a neomycin resistance gene (neo) and a triple transcriptional termination sequence (tpA). The entire neo-tpA-αSyn conditional transgene cassette is situated adjacent to the endogenous ROSA26 promoter. Following Cre-mediated excision of the neo-tpA cassette, the α-synuclein transgene is expressed from the adjacent ROSA26 promoter. ROSA26-αSyn mice were genotyped using the indicated PCR primers P1, P2 and P3. Primers P1/P3 produce a wild-type band of ~500 bp whereas primers P1/P2 produce a transgenic band of ~250 bp. Following Cre-mediated excision, primers P1/P3 can also amplify a ~1.5 kb band from the recombined transgenic allele. SA, splice acceptor; pA, polyadenylation sequence. (B) Southern blot analysis of EcoRV-digested tail genomic DNA derived from either wild-type (-/-), heterozygous (+/-) or homozygous (+/+) ROSA26-αSyn119 mice. Blots were hybridized with a [32P]-labeled DNA probe as indicated in (A). The probe detects the wild-type allele at ~11 kb and the transgenic allele at ~3.8 kb owing to an additional EcoRV site in the neo gene, as indicated. (C) Breeding strategy to produce floxed ROSA26-αSyn119+/+/TH-Cre mice and their non-floxed ROSA26-αSyn+/+ littermates at a frequency of 25% per genotype (italics). Notice that homozygous ROSA26-αSyn119+/+/TH-Cre mice (bold) were often produced with a germ line deletion (flox) of one allele (ROSA26-αSyn119+/flox/TH-Cre) at a ratio of 1:1 (+/+ to +/flox) following crossing to TH-Cre mice. (D) PCR genotyping strategy for ROSA26-αSyn119/TH-Cre mice using primers P1, P2 and P3 to distinguish the transgenic allele (Tg, ~250 bp, P1+P2), wild-type allele (WT, ~500 bp, P1+P3), or the transgenic allele following germline deletion of the neo-tpA cassette (Tgflox, ~1.5 kb, P1+P3).