To generate the PAC-Tg(SNCAA53T) transgene, the 146 kb RPCI-1 human male P1 artificial chromosome (PAC) clone 27M07, containing the entire human SNCA (synuclein, alpha (non A4 component of amyloid precursor)) gene and 34 kb of its upstream region, was modified to have the A53T human mutation associated with autosomal dominant Parkinson's disease (G>A substitution at base 420 of the cDNA sequence). This transgene was microinjected into the pronuclei of fertilized FVB/N ova. Each transgenic founder mouse was bred to FVB/N wildtype mice to generate the individual founder lines (1 and 2). Each PAC-Tg(SNCAA53T) transgenic line was independently maintained as coisogenic on the FVB/N genetic background by breeding together as homozygotes.
To generate the Snca knockout allele, a targeting vector was designed to replace exons 4 and 5 with a reverse-oriented neomycin resistance cassette (PGK-neo from the vector pPNT). The construct was electroporated into 129S6/SvEvTac-derived TC-1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and the resulting mutant mice were generated and maintained as coisogenic on the 129S6/SvEvTac genetic background.
To generate the dbl-PAC-Tg(SNCAA53T);Snca-/- strain, homozygous PAC-Tg(SNCAA53T) line 1 mice (FVB/N genetic background) were bred with mice homozygous for the Snca knockout allele (129S6/SvEvTac genetic background). Similarly, PAC-Tg(SNCAA53T) line 2 homozygotes were bred with Snca knockout mice. These two mutant strains were then intercrossed and bred to homozygosity to create the double transgenic strain. As homozygotes, dbl-PAC-Tg(SNCAA53T);Snca-/- mice were found to carry four insertions of the PAC-Tg(SNCAA53T) transgene; two for the line 1 insert (on chomsome 3) and two for the line 2 insert (on chromosome 14). This double transgenic strain was maintained on a mixed FVB/N;129S6/SvEvTac background using multiple breeding units without repeated brother-sister matings: thus the only portions of the FVB/N or 129S6/SvEvTac genomes fixed in these mice are the FVB/N regions flanking the PAC-Tg(SNCAA53T) transgene insertion sites and the 129S6/SvEvTac regions around the Snca knockout allele. The dbl-PAC-Tg(SNCAA53T);Snca-/- mice were sent to The Jackson Laboratory Repository. Upon arrival, mice were bred together to establish the colony.
