B6.Cg-Park7^tm1Shn Gpx1^tm1Ysh Prkn^tm1Shn/MgoldJ

Stock number: 023968
Product name: Parkin -/-DJ-1-/-Gpx1-/-
Research areas: Neurobiology Research, Research Tools

Description

This triple knockout strain is useful in studies of dopaminergic neuron physiology, regulation of striatal dopamine and serotonin, and Parkinson's disease.

Detailed description

In the brains of patients with Parkinson's disease, the antioxidant enzyme glutathione peroxidase 1 activity level is diminished. Mutations in human PARK7 and PARK2 resulting loss of function have been linked to types of autosomal recessive Parkinson's disease. These mice carry targeted mutations for the Park7, Gpx1 and Park2 genes. Mice that are homozygous for all 3 of the targeted mutations are viable and fertile. In triple mutant mice, 12 to 18 months of age, striatal dopamine and serotonin levels are elevated, but with normal nigral neuron numbers. Parkin^-/- DJ-1^-/- Gpx1^-/- exhibit enhanced motor behavior and are able to outperform wildtype controls in the rotarod test. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background.

Development

For the Park7 allele, a targeting vector designed by Dr. Jie Shen (Brigham & Women's Hospital, Harvard University) containing a PGK-neomycin cassette was used to disrupt exon 2. The construct was electroporated into (C57BL/6 x 129)F1 derived MKV6.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were backcrossed to C57BL/6J for 10 generations.

For the Gpx1^tm1Ysh allele, a targeting vector designed by Dr. Ye-Shih Ho (Wayne State University) containing a NEO cassette was used to disrupt exon 2. The construct was electroporated into 129 derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were crossed for approximately 6 generations to C57BL/6. Dr. Matthew Goldberg obtained the mice and backcrossed them to C57BL/6J for 3 or 4 generations. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.

For the Park2 allele, a targeting vector designed by a targeting vector designed by Dr. Jie Shen (Brigham & Women's Hospital, Harvard University) containing an in-frame EGFP coding sequence (followed by translation and transcription termination sequences and a PGK-neomycin cassette) was used to disrupt most of exon 3. The construct was electroporated into the 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were backcrossed to C57BL/6J for 10 generations.

To generate this triple mutant strain, the Park7 and Park2 knockout lines were bred and intercrossed to obtain double knockout mice. The double knockout mice were then crossed to the Gpx1 knockout mice for 2 generations, and then intercrossed to obtain mice homozygous for all 3 alleles, Parkin^-/- DJ-1^-/- Gpx1^-/- (See SNP note below). Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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. While 26 of the 27 markers throughout the genome suggested a C57BL/6 genetic background, 2 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: Prkn^tm1Shn
Name: targeted mutation 1, Jie Shen
MGI accession ID: MGI:2681404
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Prkn
Marker name: parkin RBR E3 ubiquitin protein ligase
Chromosome: 17
Strain of origin: 129S4/SvJae
Molecular note: Exon 3 was replaced in-frame by the coding sequence for EGFP followed by a PGK-neomycin cassette. RT-PCR analysis indicated that exon 2 spliced to exon 4 in transcripts thus skipping exon 3 entirely. This results in a frame shift and a premature stop codon in exon 5. Western blot analysis using antibody specific to C-terminal sequences indicated the absence of gene product.

Allele

Symbol: Park7^tm1Shn
Name: targeted mutation 1, Jie Shen
MGI accession ID: MGI:3579140
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Park7
Marker name: Parkinson disease (autosomal recessive, early onset) 7
Chromosome: 4
Strain of origin: (C57BL/6 x 129)F1
Molecular note: Exon 2 was replaced with a pgk-neo cassette. Western blot failed to detect protein in mutant mice.

Allele

Symbol: Gpx1^tm1Ysh
Name: targeted mutation 1, Ye-Shih Ho
MGI accession ID: MGI:2158814
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Gpx1
Marker name: glutathione peroxidase 1
Chromosome: 9
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: Insertion of a neomycin resistance cassette disrupted exon 2 of the gene. Northern blots of total RNA isolated from brain, heart, kidney, liver, and lung of homozygous mutant mice showed no detectable transcript of the targeted gene when probed with an a gene-specific cDNA probe.