B6;CBA-Tg(ATXN3*)84.2Cce/IbezJ

Stock number: 012705
Product name: MJD84.2
Strain synonyms: B6.SCA3-YAC-84Q
Research areas: Developmental Biology Research, Neurobiology Research

Description

These MJD84.2 transgenic mice harbor a YAC transgene that expresses a human (ATXN3 gene modified with an expanded 84 CAG repeat motif that is associated with Machado-Joseph disease in humans.

Detailed description

MJD84.2 transgenic mice (also called SCA3-YAC-84Q) harbor a YAC transgene that expresses a human ataxin 3 gene (ATXN3; also called Machado-Joseph disease [MJD], MJD1, or spinocerebellar ataxia 3 [SCA3]) modified with a human MJD/SCA3-associated 84 CAG repeat expansion [(CAG)₂CAAAAGCAGCAA(CAG)78 repeat motif (Q₃KQ₈₀), also referred to as (CAG)₈₄]. See below for comment on CAG repeat stability. These MJD84.2 transgenic mice may be useful in studying the progressive neurodegenerative processes underlying Machado-Joseph disease/spinocerebellar ataxia 3 (MJD/SCA3) pathogenesis and other polyglutamine and trinucleotide repeat disorders.

Hemizygous mice (MJD84.2) harbor two copies of the transgene at a single genomic integration site, with transgene expression levels and patterns almost identical to endogenous MJD. Transgene expression is widespread - detected in the cerebellum, cerebral cortex, heart, lung, spleen, liver and skeletal muscle. Stable transmission of the MJD1/CAG84 transgene has been demonstrated for multiple generations with a predicted frequency of about 50%.

Hemizygous mice are viable and fertile, exhibiting attenuated weight gain and a progressive neurological phenotype. The neurological phenotype is characterized by prominent gait abnormalities (~4 weeks), mild tremor, moderate hypoactivity, forelimb/hindlimb clasping (~24 weeks), inability to correct during negative geotaxis (~20 weeks), marked neuronal degeneration and mild gliosis of the dentate and pontine nerve nuclei, atrophy of the cerebellar Purkinje and molecular cell layers, and increased neuronal intranuclear inclusions (NIIs) that are ubiquitinated. With age, mice develop deficits in beam walking and gait walking assays, as well as neuronal loss in pontine nuclei and substantia nigra regions.

Homozygous mice (MJD84.2/84.2) exhibit increased severity and earlier onset of symptoms, as well as excessive grooming.

For these MJD84.2 mice, the donating investigator did not sequence the CAG repeat expansion over time. It is unknown if the CAG repeat number is subject to germline and somatic instability (whether it may expand or contract). When using lines with CAG repeat length, it is strongly recommended the CAG repeat number be quantified in all the experimental animals; all animals in all experimental groups should carry comparable CAG repeat sizes. CAG repeat sizing in mice should be done using high-resolution methods; as assays based on agarose gel electrophoresis may not provide sufficient resolution to accurately measure CAG repeat numbers. If labs do not have access to the appropriate equipment for determining CAG repeat length, CAG repeats can be evaluated on a fee-for-service basis by Laragen, Inc.

It is the experience of The Jackson Laboratory that the CAG repeat values of hemizygous MJD84.2 mice (a single genomic integration of two copies of the transgene) have been generally consistent over time, with the Laragen assay reporting ~65-78 CAG repeats [June 2020].

Development

The MJD84.2 transgenic mice (also called SCA3-YAC-84Q transgenic mice) were generated in the laboratory of Drs. Cemal K. Cemal, Mark A. Pook, and Susan Chamberlain (Imperial College, London, United Kingdom). The 250 kb human yeast artificial chromosome (YAC) 9AF12, containing the entire ~48 kb human ataxin 3 genomic locus (ATXN3; also called Machado-Joseph disease (MJD), MJD1, or spinocerebellar ataxia 3 (SCA3)) and ~35 kb of 5' and ~170 kb of 3' flanking sequences, was modified by introducing expanded CAG repeat motifs into the exon 4 region (canonical exon 10 - see note on exon numbering below) of the ATXN3 locus. The URA3 gene on the YAC was ablated in this process. A modified YAC construct with the (CAG)₂CAAAAGCAGCAA(CAG)78 repeat motif (Q₃KQ₈₀), also referred to as (CAG)₈₄, was identified and used as a source of the MJD1/CAG84 transgene. The MJD1/CAG84 transgene was microinjected into the pronuclei of (C57BL/6J x CBA/Ca)F2 oocytes, and transgenic founders were bred with C57BL/6J mice. Mice from founder line MJD84.2 were found to have a single genomic integration of two copies of the transgene. MJD84.2 transgenic mice on a "C57BL/6J" genetic background were used to cryopreserve sperm, and this sperm was sent to the laboratory of Drs. Ilya Bezprozvanny and Robert E. Hammer (University of Texas Southwestern Medical Center at Dallas). There, sperm was used to fertilize C57BL/6 oocytes to generate their colony. The resulting MJD84.2 transgenic mice on a C57BL/6 genetic background (B6.SCA3-YAC-84Q mice) were bred with wildtype mice from the colony for several generations (see SNP note below), and then sent to The Jackson Laboratory Repository. Upon arrival at The Jackson Laboratory Repository, mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

In 2011, 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. At least 13 markers on 11 chromosomes were found to be segregating, suggesting an incomplete backcross.

As of 2021, restocking of frozen sperm supplies has utilized 2-3 additional crosses to C57BL/6J. Accordingly, the 2021 SNP panel results (cohort of 2 hemizygous mice and 2 noncarriers from the colony) are much more C57BL/6J than previous results. Additionally, a Chromosome 1 SNP [~87 Mbp] was segregating for hemizygous mice but not for noncarriers - which may indicate transgene insertion [not specifically confirmed]. In 2023, SNP panel results show some transgenic mice have that SNP while some do not, and also some non-transgenic mice have that SNP as well. It remains unknown where the transgene has integrated.

Note on Exon Numbering: With respect to the human ATXN3 canonical transcript, the ~84 trinucleotide repeat is in exon 10 of the human ATXN3 locus of the transgene.
While the publication creating the MJD1/CAG84 construct [Cemal et al. 1999 Gene 236:53 (PMID:10433966)] describes the 84 trinucleotide repeat being in human ATXN3 exon 4, the publication using that MJD1/CAG84 construct to create and characterize the MJD84.2 transgenic mice [Cemal et al. 2002 Hum Mol Genet 11:1075 (PMID:11978767)] describes the 84 trinucleotide repeat being in human ATXN3 exon 10.
The human wildtype ATXN3 canonical transcript ATXN3-251 (ENST00000644486.2) shows the wildtype ~10 CAG repeat occurs in exon 10 (ENSE00003788002). The MJD52, MJD25 and CKC1 primers listed in the Cemal et al. 1999 and 2002 publication all map to the canonical transcript exon 10.

Allele

Symbol: Tg(ATXN3*)84.2Cce
Name: transgene insertion 84.2, Cemal K Cemal
MGI accession ID: MGI:3838155
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: MJD84.2; Q84; SCA3-YAC-84Q; YAC84Q
Marker symbol: Tg(ATXN3*)84.2Cce
Marker name: transgene insertion 84.2, Cemal K Cemal
Marker synonyms: MJD84.2; SCA3-YAC-84Q
Chromosome: UN
Strain of origin: (C57BL/6J x CBA/Ca)F2
Expressed genes: ATXN3,ataxin 3,human
Molecular note: A 250 kb YAC containing the intact human locus with 15 CAG trinucleotide repeats in exon 10 was modified by homologous recombination to expand the number of CAG repeats to 84. Two copies of the transgene inserted into this line with the number of CAG repeats for both copies confirmed by sequencing (a second founder line, 84.1, had insertion of 2 transgenes with variant triplets and altered repeat numbers). Transgene expression was detected in the cerebellum at endogenous levels and was also detected in the cerebral cortex, heart, and skeletal muscle.