B6N.Cg-Tg(FRG1,FRG2,DUX4*)34Maar/J

Stock number: 028012
Product name: D4Z4-12.5
Research areas: Neurobiology Research

Description

D4Z4-12.5 transgenic mice recapitulate important epigenetic and DUX4 expression attributes seen in human siblings/controls devoid of FSHD1. D4Z4-12.5 mice are a control strain for the D4Z4-2.5 transgenic strain carrying a D4Z4 genomic region from a contracted pathogenic FSHD1 allele (Stock No. 027991), and both strains are useful in studying facioscapulohumeral dystrophy.

Detailed description

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy predominantly affecting facial and upper extremity muscles. Hearing loss and retinopathy are frequently observed extramuscular features. FSHD1 is caused by contraction of the D4Z4 macrosatellite repeat to 10 or less D4Z4 units in the subtelomeric region of 4q on the 4A161 haplotype. DUX4 is more efficiently silenced with increasing D4Z4 copy number: human FSHD1 is associated with 1-10 D4Z4 repeat units, whereas the unaffected population has 11-100 D4Z4 repeat units. When expressed in skeletal muscle, the DUX4 transcription factor activates genes normally expressed in the germline (essentially inducing a stem cell program in the postmitotic muscle cell) and also suppresses several genes involved in the innate immune response.

The D4Z4-12.5 transgene has the human subtelomeric region of 4q35 (~130 kbp containing several centromeric genes and 12.5 D4Z4 repeat units [see note below] followed by the haplotype 4A161 pLAM DUX4 exon 3 [with polyA signal]). Hemizygous D4Z4-12.5 mice recapitulate important epigenetic and DUX4 expression attributes seen in human siblings/controls devoid of FSHD1; including moderate-to-high DUX4 expression levels in the germline and sufficient epigenetic repression of DUX4 in somatic tissue. Importantly, the donating investigator reports that resizing efforts in 2015 suggest that D4Z4-12.5 transgenic mice carry an array of 15.5 D4Z4 repeat units.

The phenotype listed below compares the D4Z4-2.5 transgenic mice carrying a D4Z4 genomic region from a contracted pathogenic FSHD1 allele (Stock No. 027991) with the D4Z4-12.5 transgenic mice carrying a normal sized, non-pathogenic allele (Stock No. 028012). Unless noted otherwise, hemizygous mice are described.


The fewer (2.5) D4Z4 repeat units in D4Z4-2.5 mice causes inefficient DUX4 repression in somatic tissue (bodywide hypomethylation of D4Z4) and manifests in features of human FSHD1. In contrast, D4Z4-12.5 mice have a D4Z4 repeat array (12.5) that maintains somatic tissue epigenetic silencing of DUX4 at a low enough level sufficient to preclude FSHD1 phenotype in humans.
D4Z4-2.5 mice have abundant levels of DUX4 mRNA in germ line tissues (most notably in testis), in ES cells and in early developmental stages. In D4Z4-12.5 mice, the pattern is similar but at lower levels.
In adult somatic tissue from D4Z4-2.5 mice, DUX4 mRNA expression is low and variegated in all analyzed skeletal muscles, and a variegated expression pattern of DUX4 protein in skeletal muscle nuclei is observed. In adult somatic tissue from D4Z4-12.5 mice, DUX4 transcripts are reproducibly detected only in the tibialis anterior and pectoralis muscles, whereas all other somatic tissues do not show reproducible DUX4 expression.
D4Z4-2.5 mice exhibit chromatin relaxation of the D4Z4 repeats compared to D4Z4-12.5 mice.
In both D4Z4-2.5 and D4Z4-12.5 mice, the overall morphology/histology of the limb and some head muscles appear normal. Hemizygous mice are viable and fertile. The donating investigator also reports that homozygous mice are viable and fertile, and that the number of D4Z4 repeats within each line stays the same through several generations of breeding (i.e., the D4Z4 repeat unit is stable).
Approximately half of the D4Z4-2.5 mice develop eye abnormalities (keratitis) around 8-12 weeks of age.

Development

The D4Z4-12.5 transgene was designed by Dr. Silvere M. van der Maarel (Leiden University Medical Center, The Netherlands) with the non-permissive human facioscapulohumeral dystrophy (FSHD) allele using co-injection of two overlapping PAC clones.
Specifically, the 133 kbp kbp PAC clone 226K22 (Genbank accession number AF146191) contained the complete genomic sequences of the human FRG1 gene, TUBB7P pseudogene and FRG2 gene.
The 110 kbp RPCI-6 library 202J3 PAC clone contained a genomic fragment extending from the inverted D4Z4 repeat unit (D4S2463; ~40 kbp proximal to D4Z4) through the D4Z4 repeat region. This D4Z4 region has at least twelve-and-a-half copies (see note below) of the D4Z4 macrosatellite repeat units (each unit has exons 1-2 of the DUX4 retrogene; with exon 1 encoding a double homeobox transcription factor), the region telomeric to the last repeat (called pLAM) contains the DUX4 exon 3 (harboring the FSHD-permissive haplotype 4A161 polyA signal) and lacks the more downstream DUX4 exons 4-7. Important note: the donating investigator reports that resizing efforts in 2015 suggest that D4Z4-12.5 transgenic mice carry an array of 15.5 D4Z4 repeat units.
The two PAC transgenes were co-microinjected into the pronuclei of fertilized oocytes of B6CBAF1/J mice (Charles River Laboratories), and then founder animals were bred to C57BL/6NCrl mice for germline transmission. Only one founder line (K22J3-34) was generated in which the two PAC transgenes recombined as a single overlapping ~183 kbp integration (on chromosome 2). The D4Z4-12.5 transgenic colony was then bred with C57BL/6Jico (C57BL/6NCrl) wildtype mice for at least 20 generations prior to sending black male mice to The Jackson Laboratory Repository in 2015. Upon arrival, males were used to cryopreserve sperm. To establish the living D4Z4-2.5 mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).

Allele

Symbol: Tg(FRG1,FRG2,DUX4*)34Maar
Name: transgene insertion 34, Silvere van der Maarel
MGI accession ID: MGI:5660198
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(FRG1,FRG2,DUX4*)34Maar
Marker name: transgene insertion 34, Silvere van der Maarel
Chromosome: 2
Strain of origin: (C57BL/6J x CBA/J)F1
Expressed genes: FRG1,FSHD region gene 1,human; FRG2,FSHD region gene 2,human; DUX4,double homeobox 4,human
Site of expression: DUX4 transcripts could only be reproducibly detected in the tibialis anterior and pectoralis muscles, whereas all other somatic tissues did not show reproducible DUX4 expression.
Molecular note: The transgene contains two co-injected PAC clones. PAC clone 226K22 contains the complete genomic sequences of the human FRG1 gene, TUBB7P pseudogene and FRG2 gene. PAC clone 202J3 contains a genomic fragment extending from the inverted D4Z4 repeat unit through the D4Z4 repeat region. This D4Z4 region has twelve-and-a-half copies of the D4Z4 macrosatellite repeat units (each unit has exons 1-2 of the DUX4 retrogene; with exon 1 encoding a double homeobox transcription factor), the region telomeric to the last repeat (called pLAM) contains the DUX4 exon 3 (harboring the FSHD-permissive haplotype 4A161 polyA signal) and lacks the more downstream DUX4 exons 4-7 (per primary investigator). The primary investigator reports that resizing efforts in 2015 suggest that D4Z4-12.5 transgenic mice carry an array of 15.5 D4Z4 repeat units. The two PAC transgenes recombined as a single overlapping ~183 kbp integration on chromosome 2. DUX4 mRNA was detected in germline tissues. In analyzed somatic tissues, transcripts are detected only in the tibialis anterior and pectoralis muscles, whereas all other somatic tissues do not show reproducible DUX4 expression.