FVB;129S6-Tg(SNCA*A53T)1Nbm Snca^tm1Nbm Tg(SNCA*A53T)2Nbm/J

Stock number: 010799
Product name: Snca^-; dbl-PAC-Tg(SNCA^A53T)
Strain synonyms: dbl-PAC-Tg(SNCAA53T);Snca-/-
Research areas: Internal/Organ Research, Neurobiology Research, Research Tools

Description

These double transgenic homozygous mice (dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice) harbor a Snca knock-out allele and two independently inserted transgenes encoding the human A53T-mutant α-synuclein associated with autosomal dominant Parkinson's disease. The very early gastrointestinal dysfunction in the absence of major central nervous system pathology in dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice mimics what is seen early in human Parkinson's disease, when enteric nervous system dysfunction may precede the more classical motor symptoms by years to decades.

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

These double transgenic homozygous mice (dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice) are viable and fertile, harboring a Snca knock-out allele and two independently inserted transgenes encoding the human A53T-mutant α-synuclein associated with autosomal dominant Parkinson's disease. As homozygotes, expression of endogenous mouse α-synuclein is abolished and replaced by α-synuclein*A53T from the insertions of the PAC-Tg(SNCA^A53T). While brain RNA expression of SNCA^A53T is ~10-fold greater compared to normal endogenous mouse α-synuclein, protein levels are only 1 to 1.5-fold greater. In colon however, both RNA and protein expression of SNCA^A53T are ~80-fold greater than normal endogenous mouse α-synuclein. By three months of age, dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice show robust abnormalities in enteric nervous system function (impaired colonic motility [males only] and prolonged gut transit time). Significant α-synuclein*A53T aggregation is observed in colonic myenteric plexus, while no widespread aggregation in brain is reported. Subtle motor behavior abnormalities develop between 6-12 months. No detectable autonomic abnormalities, olfactory dysfunction, dopaminergic deficits, Lewy body inclusions or neurodegeneration are associated with α-synuclein*A53T expression in these double transgenic mice. The very early gastrointestinal dysfunction in the absence of major central nervous system pathology in dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice mimics what is seen early in human Parkinson's disease, when enteric nervous system dysfunction may precede the more classical motor symptoms by years to decades.

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

To generate the PAC-Tg(SNCA^A53T) 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(SNCA^A53T) transgenic line was independently maintained as coisogenic on the FVB/N genetic background by breeding together as homozygotes.

To generate the Snca knock-out 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(SNCA^A53T);Snca^-/- strain, homozygous PAC-Tg(SNCA^A53T) line 1 mice (FVB/N genetic background) were bred with mice homozygous for the Snca knock-out allele (129S6/SvEvTac genetic background). Similarly, PAC-Tg(SNCA^A53T) line 2 homozygotes were bred with Snca knock-out mice. These two mutant strains were then intercrossed and bred to homozygosity to create the double transgenic strain. In 2010, homozygous mice [dbl-PAC-Tg(SNCA^A53T);Snca^-/-] were originally reported to carry four insertions of the PAC-Tg(SNCA^A53T) transgene; two for the line 1 insert on chromosome 3 and two for the line 2 insert on chromosome 14 [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)]. 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(SNCA^A53T) transgene insertion sites and the 129S6/SvEvTac regions around the Snca knock-out allele. The dbl-PAC-Tg(SNCA^A53T);Snca^-/- mice were sent to The Jackson Laboratory Repository. Upon arrival, mice were bred together to establish the colony.

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: 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.