B6(Cg)-Gt(ROSA)26Sor^{tm1.1(DUX4*)Plj}/J

Stock number: 028710
Product name: FLExDUX4
Strain synonyms: Dux4-fl
Research areas: Neurobiology Research, Research Tools

Description

FLExDUX4 mice conditionally express human DUX4 following Cre-mediated recombination. This strain may be useful for studying facioscapulohumeral muscular dystrophy (FSHD).

Detailed description

FLExDUX4 mice were created using a cre-dependent one-way genetic switch (FLEx) system. Homozygotes mice carrying this DUX4 conditional allele are viable and fertile. Two sets of incompatible outward facing recombination sites (loxP and lox511) flank an inverted human double homeobox 4 (DUX4) sequence, including exons 1-3 and both introns. The DUX4 gene encodes several alternative mRNA splicing variants. The hereditary muscle disorder, facioscapulohumeral muscular dystrophy (FSHD) is caused by the toxic gain-of-function of the DUX4-full-length (DUX4-fl) mRNA isoform. The DUX4-fl mRNA, which encodes a paired homeobox domain transcription factor, is typically not expressed in healthy muscle. However, in FSHD, the rare expression of DUX4-fl (in less than 1% of muscle fibers) initiates a pathogenic cascade of events including apoptosis, differentiation defects, muscle atrophy, and susceptibility to oxidative stress. Overall, FSHD is characterized by a slowly progressing muscular dystrophy that predominantly affects the skeletal muscles of the face, scapula, and upper arms but can affect muscles of the abdomen, hip girdle, and lower legs with ~20% of patients ultimately losing ambulation.

The DUX4 promoter drives expression of two short non-pathogenic isoforms (DUX4-s) and a longer cytotoxic isoform (DUX4-fl). This strain contains 4 point mutation in the 5’ splicing donor sites for the two DUX4-s mRNAs, abolishing expression of the short isoforms and only generating the pathogenic DUX4-fl mRNA isoform.

Because this construct was targeted for insertion into the Gt(ROSA)26Sor locus, DUX4-fl expression is determined by which tissue(s) express Cre recombinase. When bred to mice that express Cre recombinase, the resulting offspring will have the loxP or lox511 sites recombined, resulting in the inversion of the human DUX4-fl sequence, ending in a sense orientation.

 

Hemizygous and homozygous mice have low level DUX4-fl expression in the absence of Cre Recombinase. These mice exhibit alopecia, soft stool, inflammation, and muscle weakness. Homozygous are more affected, as are males compared to females.

When bred to STOCK Tg(ACTA1-cre/Esr1*)2Kesr/J mice (Stock No. 025750), resulting offspring express slightly more DUX4-fl than the FLExDUX4 hemizygous mice prior to tamoxifen induction (TMX). One TMX injection of 5 mg/kg leads to a useful mildly progressive FSHD model, while 2 injections of 10mg/kg leads to a more severe model.

Of note, the donating investigator reports that when bred to Sox2-Cre mice (Stock No. 008454), resulting offspring are embryonic lethal. When bred to ACTA1-rtTA, tetO-cre mice (Stock No. 012433), resulting offspring are embryonic lethal. When bred to ACTA1-Cre mice (Stock No. 006149), resulting offspring are still-born. When bred to UBC-CreERT2 mice (Stock No. 008085), without tamoxifen induction, offspring begin to show a severe neurological phenotype around 5 weeks of age, perhaps due to leaky DUX4 expression in the brain.

Development

The FLExDUX4 targeting vector was designed with (from 5’ to 3’) a loxP site, a frt-flanked neomycin resistance gene, a lox511 site, an inverted human double homeobox 4 (DUX4) gene with a polyadenylation sequence, and another loxP site and lox511 site, both in opposite orientation to the 5’ sites. The DUX4 gene contains 4 silent point mutations in exon 1 that prevent alternative splicing of a truncated isoform, DUX4-s, while maintaining the DUX4-fl ORF. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of C57BL/6-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into B6(Cg)-Tyr^c-2J/J blastocysts, and resulting chimeric males were bred to C57BL/6J females. Subsequently, mice were bred to mice expressing Flp recombinase on a C57BL/6N background to remove the neo cassette. Offspring were bred together to remove the Flp-expressing transgene and a homozygous colony of FLExDUX4 mice was created. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 upon arrival. While the 43 markers throughout the genome suggested a C57BL/6 genetic background, 5 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/6N;C57BL/6J genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(DUX4*)Plj}
Name: targeted mutation 1.1, Peter Jones
MGI accession ID: MGI:5707276
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence; Humanized sequence
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: C57BL/6
Expressed genes: DUX4,double homeobox 4,human
Molecular note: The targeting vector was designed with (from 5 to 3) a loxP site, an FRT-flanked neomycin resistance gene, a lox511 site, an inverted human double homeobox 4 (DUX4) gene with a polyadenylation sequence, and another loxP site and lox511 site, both in opposite orientation to the 5 sites. The DUX4 gene contains 4 silent point mutations in exon 1 that prevent alternative splicing of a truncated isoform, DUX4-s, while maintaining the DUX4-fl ORF. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. Flp-mediated recombination removed the FRT-flanked neo cassette.