STOCK Gba^tm1Rlp Tg(SNCA*A53T)1Nbm Snca^tm1Nbm Tg(SNCA*A53T)2Nbm/Mmjax

Stock number: 027227
Product name: Gba^L444P; Snca^-; dbl-PAC-Tg(SNCA^A53T)
Research areas: Internal/Organ Research, Neurobiology Research

Description

The dbl-PAC-Tg(SNCA^A53T);Snca^-/-; Gba^L444P/+ mice harbor a Snca knock-out allele, two transgenes encoding the human A53T-mutant alpha-synuclein associated with autosomal dominant Parkinson's disease and the L444P mutation in the Gba (glucosidase, beta, acid) gene associated with the alpha-synucleinopathy, Gaucher disease. Combining these two mouse models of Parkinson's and Gaucher disease results in an exacerbation of the motor and gastrointestinal deficits found in the Parkinson's A53T model alone.

See Detailed Description section for updated information regarding the transgene insertion sites, copy number and complexity (genomic duplication/rearrangement, co-integration with multiple genomic fragments) [July 2022].

Detailed description

The dbl-PAC-Tg(SNCA^A53T);Snca^-/-; Gba^L444P/+ mice harbor a Snca knock-out allele, two independently inserted transgenes encoding the human A53T-mutant alpha-synuclein associated with autosomal dominant Parkinson's disease and the L444P mutation in the Gba (glucosidase, beta, acid) gene associated with the alpha-synucleinopathy, Gaucher disease. In mice homozygous for the two transgenes and the Snca KO, expression of endogenous mouse alpha-synuclein is abolished and replaced by alpha-synuclein*A53T from the four total insertions of the PAC-Tg(SNCA^A53T) resulting in a Parkinson's phenotype (see Jackson Laboratory Stock No. 010799). The addition of the heterozygous Gaucher mutation, L444P, reduces glucocerebrosidase (GBA) activity by 40%, reduces SNCA degradation, and results in the accumulation SNCA protein in culture. The enzyme GBA is involved in lysosomal protein degradation and thus in the lysosomal-autophagy pathway mainly responsible for SNCA degradation. Heterozygous carriers of Gaucher disease have an increased risk for Parkinson's disease. 14 to 15 month old mice exhibit an enteric nervous system dysfunction characterized by constipation and reduced whole gut transit time (males only). Motor activity is impaired as well as exploratory behavior, and endurance and coordination on the rotarod. Accumulation of human pSer129 SNCA is observed earlier (15 months) than in the A53T mice, although there is no increase in absolute SNCA levels. Combining these two mouse models of Parkinson's and Gaucher disease results in an exacerbation of the motor and gastrointestinal deficits found in the Parkinson's A53T model alone.

Transgene insertion sites, copy number and complexity (genomic duplication/rearrangement, co-integration with multiple genomic fragments):
 Summarizing the updated information for Stock No. 010799 (July 2022): The PAC-Tg(SNCA^A53T) line 1 has ~6 copies inserted on Chr3 (~126.4Mb) with genomic duplication/rearrangement and the PAC-Tg(SNCA^A53T) line 2 has ~3 copies inserted on Chr14 (~111.9Mb) that co-integrated with multiple genomic fragments containing several loci. The specific details are further below. Regarding the copy numbers, mice homozygous for both line 1 and line 2 would have a total of ~18 copies of SNCA^A53T. Importantly, ddPCR results for Stock Nos. 010799 and 027227 [MMRRC_037633-JAX] indicate the same copy numbers for both strains.
 In 2010, the donating laboratory reported their FISH results indicated PAC-Tg(SNCA^A53T) transgenes were inserted on mouse chromosomes 3 and 14 - denoted as PAC-Tg(SNCA^A53T) line 1 and PAC-Tg(SNCA^A53T) line 2, respectively. In support of that finding, FISH/qPCR investigation by The Jackson Laboratory in 2010 showed homozygous dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice exhibited fluorescent signals on both homologs of Chr3 and on both homologs of Chr14, and estimated homozygous dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice carried four insertions of the PAC-Tg(SNCA^A53T) transgene: two for the line 1 insert on Chr3 and two for the line 2 insert on Chr14.
 In July 2022, advanced quality control analysis commissioned/performed by The Jackson Laboratory (targeted locus amplification, sequencing, ddPCR) for Stock No. 010799 was concluded and identified the precise PAC-Tg(SNCA^A53T) transgene insertion coordinates, illustrated the complex nature of the transgene insertion sites and updated the estimated copy number for each.
 A) The PAC-Tg(SNCA^A53T) line 1 transgene integration site is Chr3:126,388,132-126,412,554 (genome build GRCm38/mm10). The updated copy number estimate for line 1 is ~6 copies as hemizygote (~12 copies as homozygote). The Chr3 integration is accompanied by a 24 kb genomic duplication and genomic rearrangements within and near the duplicated region. The duplicated region contains exons 1 and 2 of the Arsj locus.
 B) The PAC-Tg(SNCA^A53T) line 2 transgene integration site is Chr14:111,885,431-111,915,270 (genome build GRCm38/mm10). The updated copy number estimate for line 2 is ~3 copies as hemizygote (~6 copies as homozygote). Multiple genomic fragments from chromosomes 11, 13 and 14 have co-integrated with the transgene. It was not determined if each fragment represents a genomic translocation or duplication. Listed below are each fragment size and the predicted RefSeq genes within each fragment (asterisk indicates partial locus missing its 5' region [* before gene name] or missing its 3' region [* after gene name]; location coordinates correspond to genome build GRCm38/mm10).
 : Chr11:59,590,364-59,604,395 (14 kb, no genes annotated here)
 : Chr11:~83,546,640-83,591,121 (44.5 kb, Ccl9, E230016K23Rik*, *Ccl6)
 : Chr13:74,235,894-74,258,296 (22.3 kb, *Ahr* [only one exon])
 : Chr13:74,292,249-74,307,809 (15.5 kb, *Pdcd6)
 : Chr13:100,440,526-100,470,965 (30.4 kb, Naip1*, *Gtf2h2)
 : Chr13:100,486,415-100,564,602 (78.1 kb, Gtf2h2*, Ocln)
 : Chr13:100,640,831-100,707,238 (66.4 kb, Rad17*, Taf9, Ak6, Ccdc125, *Cdk7)
 : Chr13:100,828,312-100,915,974 (87.7 kb, Slc30a5*, Gm29502*, Gm41040)
 : Chr14:45,556,508-45,638,760 (82.2 kb, *4930527F14Rik, Gm15601, Gm49124, Gm41145, *Ddhd1 [all but its first exon])

Development

The PAC-Tg(SNCA^A53T) transgene was designed using 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 modified to have the A53T human mutation. 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). [see Detailed Description section for 2022 updated information regarding the transgene insertion sites, copy number and complexity (genomic duplication/rearrangement, co-integration with multiple genomic fragments)].

The targeting vector for the Snca knock-out allele was designed to replace exons 4 and 5 with a reverse-oriented neomycin resistance cassette. 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 chimeric mice were crossed to 129S6/SvEvTac.

The transgenic dbl-PAC-Tg(SNCA^A53T) strains and the Snca-/- strain were combined to form a homozygous double transgenic, homozygous Scna KO strain maintained on a mixed FVB/N;129S6/SvEvTac background. The triple mutant is distributed by The Jackson Laboratory as Stock No. 010799).

The targeting vector for the Gba L444P knockin allele was designed to insert a proline to leucine substitution mutation at amino acid position 444 in exon 10. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and the resulting chimeric mice were crossed to C57BL/6. The Gba^L444P mutant is distributed by MMRRC as Stock No. 000117-UNC. The triple SCNA mutant was crossed to Gba^L444P mice and a colony was generated by random intrastrain matings that is homozygous for both transgenes and the Scna KO and heterozygous for the Gba^L444P allele.

Allele

Symbol: Tg(SNCA*A53T)1Nbm
Name: transgene insertion 1, Robert L Nussbaum
MGI accession ID: MGI:4412061
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(SNCA*A53T)1Nbm
Marker name: transgene insertion 1, Robert L Nussbaum
Chromosome: 3
Strain of origin: FVB/N
Expressed genes: SNCA,synuclein alpha,human
Molecular note: 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. The transgene integration site is Chr3:126,388,132-126,412,554 (genome build GRCm38/mm10). The copy number estimate for line 1 is ~6 copies as hemizygote (~12 copies as homozygote). The Chr3 integration is accompanied by a 24 kb genomic duplication and genomic rearrangements within and near the duplicated region. The duplicated region contains exons 1 and 2 of the Arsj locus.

Allele

Symbol: Tg(SNCA*A53T)2Nbm
Name: transgene insertion 2, Robert L Nussbaum
MGI accession ID: MGI:4412066
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(SNCA*A53T)2Nbm
Marker name: transgene insertion 2, Robert L Nussbaum
Chromosome: 14
Strain of origin: FVB/N
Expressed genes: SNCA,synuclein alpha,human
Molecular note: 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. The transgene integration site is Chr14:111,885,431-111,915,270 (genome build GRCm38/mm10). The updated copy number estimate for line 2 is ~3 copies as hemizygote (~6 copies as homozygote). Multiple genomic fragments from chromosomes 11, 13 and 14 have co-integrated with the transgene. It is not known if these are translocations or duplications.

Allele

Symbol: Gba^tm1Rlp
Name: targeted mutation 1, Richard L Proia
MGI accession ID: MGI:2180202
Generation method: Targeted
Attributes: Hypomorph; Humanized sequence
Marker symbol: Gba
Marker name: glucosidase, beta, acid
Chromosome: 3
Strain of origin: 129S4/SvJae
Molecular note: A proline to leucine substitution mutation at amino acid position 444 is associated with Gaucher disease type 3 in humans. This mutation was introduced in mice using a single insertion mutagenesis procedure. The level of gene expression in brain of mutant mice is similar to wild-type as determined by Northern blot analysis, but enzyme assays with liver, brain, and skin extracts demonstrate that enzyme activity is 20% that of wild-type.

Allele

Symbol: Snca^tm1Nbm
Name: targeted mutation 1, Robert L Nussbaum
MGI accession ID: MGI:2389489
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Snca
Marker name: synuclein, alpha
Chromosome: 6
Strain of origin: 129S6/SvEvTac
Molecular note: Exons 4 and 5 were replaced with a neomycin selection cassette inserted by homologous recombination. While the separation of Western blot results by 2D-PAGE showed various isoforms in total brain extract obtained from wild-type mice, protein was undetected in samples obtained from homozygous mutant mice.