B6.Cg-Polg^tm1Prol Prkn^tm1Shn/J

Stock number: 036670
Product name: PolgA^D257A; Parkin-
Strain synonyms: Parkin KO x Polg mutator, Polg mutator ; PKO
Research areas: Cell Biology Research, Metabolism Research, Neurobiology Research

Description

The double mutant Stock No. 036670 was created by breeding PolgA^D257Aneo males from Stock No. 017341 (B6.129S7(Cg)-Polg^tm1Prol/J) to Parkin knock-out females from Stock No. 006582 (B6.129S4-Prkn^tm1Shn/J).

The Polg^D257A mitochondrial mutator mice have a D257A mutation in the N-terminal "proofreading" exonuclease domain of the DNA polymerase γ gene (Polg), rendering the expressed mutant protein devoid of polymerase proofreading function in mitochondria. Homozygous Polg^D257A mice develop a premature aging phenotype.

Prkn (parkin RBR E3 ubiquitin protein ligase) knock-out mice develop increased extracellular dopamine concentration in the striatum, dysfunctional nigrostriatal pathways, and dysfunctional mitochondria. Double mutant mice gain less weight than either controls or the Polg^D257A homozygotes. This strain maybe useful for studies of mitochondrial dysfunction.

Detailed description

Prkn (parkin RBR E3 ubiquitin protein ligase) encodes a component of the E3 ubiquitin ligase complex and is involved in proteasomal degradation and autophagy of damaged mitochondria. Mutations in Prkn are associated with Parkinson disease, specifically the exon 3 deletion mutation is most common in human autosomal recessive juvenile parkinsonism (AR-JP; Parkinson disease 2; PARK2) patients. Parkin knockout mice have exon 3 replaced by a mostly nonfunctional GFP reporter. Mice homozygous for the Prkn mutation have increased extracellular dopamine concentration in the striatum, decreased body size, impaired coordination (rotorod) and exhibit mitochondrial dysfunction and impaired protection from oxidative stress.

Polg (polymerase (DNA directed), gamma) encodes a catalytic subunit of mitochondrial DNA polymerase. Mutations in Polg are associated with several mitochondrial diseases. Polg^D257A mice carry a D257A mutation in the N-terminal "proofreading" exonuclease domain of the DNA polymerase γ gene (Polg), rendering the expressed mutant protein devoid of polymerase proofreading function in mitochondria. Homozygous Polg^D257A mice exhibit ~2500-fold increased rate of mtDNA mutation compared to wild-type mice. Homozygous mice grossly demonstrate a premature aging phenotype beginning at ~nine months of age with alopecia, graying hair, impaired mobility and kyphosis, and die prematurely by ~13-15 months of age with severe anemia.

When Polg^D257A mice are bred with Parkn knockout mice the double mutant offspring fail to gain weight as compared to controls and homozygous Polg^D257A mice. The mutations do not effect organ weight. Further characterization of the double mutant  is underway and will be added as it becomes available.

Of note, the individual mutations are available as B6.129S7(Cg)-Polg^tm1Prol/J (Stock No. 017341) and B6.129S4-Prkn^tm1Shn/J (Stock No. 006582).

 

Development

The double mutant Stock No. 036670 was created by breeding PolgA^D257Aneo males from Stock No. 017341 (B6.129S7(Cg)-Polg^tm1Prol/J) to Parkin knock-out females from Stock No. 006582 (B6.129S4-Prkn^tm1Shn/J).

To create the Polg targeting vector, Dr. Tomas A. Prolla (University of Wisconsin, Madison) isolated a mouse DNA polymerase γ (Polg) DNA sequence and used site-directed mutagenesis to introduce an AC-->CT two-base substitution corresponding to positions 1054-1055 in exon 3. The targeting vector also had a loxP-flanked PGK-neo cassette inserted into intron 3 of Polg on chromosome 7. The point mutations create a D257A amino acid substitution in the conserved N-terminal "proofreading" exonuclease domain II of Polg. This targeting construct was electroporated into 129S7/SvEvBrd-Hprt^b-m2-derived AB2.2 embryonic stem (ES) cells, and correctly targeted ES cells were injected into blastocysts. Chimeric males were bred to C57BL/6J females. The PolgA^D257Aneo mice were then bred with CMV-Cre transgenic mice (on a mixed 129/ICR genetic background) to remove the floxed PGK-neo cassette. The resulting mice harboring the PolgA^D257A allele (also called Polg^D257A, Polg^mut, or Polg^A) were subsequently backcrossed 16 generations to C57BL/6J wildtype mice (and the CMV-Cre transgene was removed) prior to sending males to The Jackson Laboratory in 2012 as Stock No. 017341.

The Prkn knock-out targeting vector was designed by Dr. Jie Shen (Harvard) to replace most of exon 3 of Prkn on chromosome 17 with the in-frame EGFP coding sequence (followed by translation and transcription termination sequences and a PGK-neomycin cassette). The mutation results in a frame shift and a premature stop codon in exon 5. While EGFP transcripts are present, little parkin-EGFP fusion protein is detectable by Western analysis. The construct was electroporated into the 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The donating investigator reported that the resulting mutant mice were backcrossed to C57BL/6 inbred mice for more than 20 generations prior to sending males to The Jackson Laboratory in 2007 as Stock No. 006582.

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: Polg^tm1Prol
Name: targeted mutation 1, Tomas A Prolla
MGI accession ID: MGI:3583920
Generation method: Targeted
Attributes: Not Specified
Marker symbol: Polg
Marker name: polymerase (DNA directed), gamma
Chromosome: 7
Strain of origin: 129S7/SvEvBrd-Hprt^b-m2
Molecular note: A targeting construct was designed to introduce an AC to CT two-base substitution in positions 1054 and 1055, resulting in a critical residue substitution in the conserved exonuclease domain (D257A).