Mice carrying SncaΔflox and ROSA26-stop-lacZ reporter (Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5037-42) loci linked at the same chromosome as the result of natural crossover were selected from a pool of animals generated during crossbreeding of two mouse lines (both on C57BL/6 background), each bearing one of these modified loci.
For the Sncatm1.2Vlb allele, a targeting vector containing a FRT site-flanked NEO cassette and a loxP site was inserted downstream of exon II (the first coding exon) of the targeted Snca gene, and another loxP site was inserted upstream of exon II. This construct was electroporated into C57BL/6N-Atm1Brd-derived JM8A3.N1 embryonic stem (ES) cells. The resulting chimeric animals were bred to C57BL/6J mice for 2 generations and then crossed to transgenic mice (on a congenic C57BL/6 genetic background) expressing FLP recombinase under the control of the human ACTB promoter, generating SncafloxΔneo mice. The FLP recombinase mediated excision of the FRT flanked NEO cassette and a 400 bp fragment encompassing a simple repeat region does not affect Snca locus expression. The SncafloxΔneo mice that retained the floxed exon II were bred to C57BL/6J mice for 2 generations to remove the FLP recombinase transgene, before being intercrossed to generate homozygotes. These homozygous SncafloxΔneo/floxΔneo mice were then bred to transgenic mice (on the C57BL/6 background) expressing Cre recombinase under the control of the CMV promoter (see, for example, Stock No. 006054) to excise the floxed exon II and flanking sequences. The resulting knock-out SncaΔflox mice were then bred to C57BL/6J to remove the Cre recombinase transgene and further backcrossed with C57BL/6J mice for more than 4 generations.
For the Gt(ROSA)26Sortm1Sho, the R26R targeting construct was created by subcloning the pROSA26-1 vector, and inserting a splice acceptor sequence (identical to the one used in the original gene trap allele), a loxP-flanked STOP sequence (containing a neo cassette and triple polyadenylation (STOP) sequence), a lacZ gene, and a polyadenylation sequence at a unique site approximately 300-bp 5' of the original gene trap integration site. Transcriptional read-through is prevented with the use of a triple polyadenylation (STOP) sequence at the 3' end of the neo expression cassette. The construct was electroporated into 129S4/SvJaeSor derived AK7 embryonic stem (ES) cells. (see, for example, Stock No. 003474)
The single mutant lines were crossed to generate double mutant animals. The mice were then bred to select for these SncaΔflox ROSA26-stop-lacZ reporter mice carrying both mutant alleles on the same chromosome.
The mice were then bred to homozygosity for both alleles.
Upon arrival, embryos were cryopreserved.
A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Two of the 27 markers throughout the genome were segregating, suggesting an incomplete backcross. One of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
