STOCK Snca^tm1.2Vlb Gt(ROSA)26Sor^tm1Sho/J

Stock number: 028560
Product name: Snca delta flox ("clean KO") & Rosa26-stop-lacZ
Research areas: Neurobiology Research, Research Tools

Description

These Snca^Δflox Rosa26-stop-lacZ reporter mice carry a knock out allele of the Snca gene and the Gt(ROSA)26Sor^tm1Sho Cre reporter allele. This strain may be useful for generating mutants in which Cre recombinase efficiency can be monitored for applications related to neurodegenerative synucleinopathies, such as Parkinson's disease and multiple system atrophy.

Detailed description

These mice carry the Snca^Δflox allele (028559) and the Rosa26-stop-lacZ reporter allele (003504). The "Snca^Δflox" knock-out allele contains a 1164 bp fragment deletion (including the first coding exon (exon II) and adjacent flanking sequence) of the Snca (synuclein, alpha) gene. The Gt(ROSA)26Sor^tm1Sho allele contains a loxP-flanked DNA STOP sequence preventing expression of the downstream lacZ gene. When crossed with a cre recombinase expressing strain, the STOP sequence is removed and lacZ is expressed in cells/tissues where cre is expressed.

The Rosa26-stop-lacZ reporter allele in this double mutant line allows for the monitoring of Cre recombination efficiency when crossed to mice carrying a floxed Snca allele (see 025636). Of note, because the Snca and Rosa26 loci are located on chromosome 6, approximately 50 Mb apart, there is a possibility that recombination can separate the mutant alleles during breeding. Mice that are homozygous for these alleles are viable and fertile.

Mutations in human SNCA that result in aberrant polymerization and fibrillar aggregations are associated with neurodegenerative synucleinopathies, such as Parkinson's disease and multiple system atrophy.

Development

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 Snca^tm1.2Vlb 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-A^tm1Brd-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 Snca^floxΔneo 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 Snca^floxΔneo 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 Snca^floxΔneo/^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 Snca^Δflox 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)26Sor^tm1Sho, 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 Snca^Δflox 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.

Allele

Symbol: Gt(ROSA)26Sor^tm1Sho
Name: targeted mutation 1, Stuart Orkin
MGI accession ID: MGI:1861931
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: 129S4/SvJaeSor
Expressed genes: Bgeo,fusion of beta-galactosidase and neomycin phosphotransferase genes,E. coli
Site of expression: when crossed to a cre recombinase-expressing strain, lacZ expression is observed in the cre-expressing tissues; this is similar to Gt(ROSA)26Sor^tm1Sor but has one copy of the loxP-flanked reporter gene
Molecular note: A targeting vector was designed to insert floxed (flanked by loxP) phosphoglycerate kinase-1-cytosine deaminase/phosphoglycerate kinase-1-puromycin (PGK-CD/PGK-Puro) and stop sequences between the splicing acceptor (SA) and the beta-gal-neomycin phosphotransferase fusion gene (beta-geo) sequences of the proviral ROSA 26 locus. Correctly targeted, this resulted in ES cells that were Puro resistant but G418 susceptible, and did not express beta-gal.
General note: Mice carrying this mutation are considered to be reporter strains, with successful Cre excision indicated by beta-gal expression in Cre-expressing tissues.

Allele

Symbol: Snca^tm1.2Vlb
Name: targeted mutation 1.2, Vladimir L Buchman
MGI accession ID: MGI:5700504
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Snca
Marker name: synuclein, alpha
Chromosome: 6
Strain of origin: C57BL/6N-A^tm1Brd
Molecular note: A targeting vector containing a FRT site-flanked neo cassette and a loxP site was inserted downstream of exon 2 (the first coding exon) of the targeted gene, and another loxP site was inserted upstream of exon 2. Flp-mediated recombination removed the FRT-flanked neo cassette leaving exon 2 floxed. Germline, Cre-mediated recombination removed exon 2. Western blot analysis of neural tissues confirmed that no protein is expressed.