B6(Cg)-Fus^tm1.1Emcf/J

Stock number: 036312
Product name: B6 FUS-Δ14
Research areas: Neurobiology Research

Description

FUS Delta14 knock-in mice have a frameshift mutation introduced into exon 14 of the Fus gene, and replacement of exon 15 with the human homologue, resulting in expression of physiological levels of "humanized" mutant protein from the endogenous locus. Heterozygous FUS Delta14 mice exhibit progressive, adult-onset motor neuron degeneration and denervation of neuromuscular junctions. These mice may be useful in the study of amyotrophic lateral sclerosis (ALS).

Detailed description

The fused in sarcoma (Fus) gene encodes a part of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex which is involved in pre-mRNA splicing and the export of fully processed mRNA to the cytoplasm. Mutations in Fus are responsible for ~5% of familial case of amyotrophic lateral sclerosis (ALS), and many of these mutations occur near the final exon (exon 15).

The FUSΔ14 knock-in allele has an ALS pathogenic point mutation in exon 14, and a replacement of the endogenous exon 15 with the human exon 15 homolog. This results in skipping of exon 14 during splicing and out-of-frame translation of exon 15, recapitulating a novel frame-shifted C-terminus, as found in the FUSΔ14 patient mutation. Western blotting using an N-terminal antibody indicates that FUSΔ14 mice express comparable levels of total FUS protein, while using an antibody against the C-terminus reveals FUSΔ14 express approximately half the amount of wildtype FUS protein. The mutant FUSΔ14 protein distribution is predominantly diffuse in the nucleus and cytoplasm, with some perinuclear accumulations. Heterozygotes exhibit a 15% reduction in motor unit numbers at 18 months of age, and show progressive, age-related motor impairment, which is apparent at 12 and 15 months in the form of hindlimb errors. Gait analysis at 18 months shows FUSΔ14 mice have altered rear stride pattern (not length) (Devoy et al., 2017). Otherwise, heterozygous mice are viable and fertile.

The donating investigator reports that on the C57BL/6J background, homozygotes die shortly before or at the time of parturition. However, on a mixed B6;DBA/2 background, most homozygotes survive to at least 3 months of age.

Development

The FUS Delta14 knock-in allele (also called FUSΔ14) was created in the laboratory of Dr. Elizabeth M.C. Fisher (University College London) to have a human truncation mutation inserted into exon 14 of the fused in sarcoma locus (Fus) on chromosome 7, leading to a truncated protein with a short frame-shifted C-terminus. The specific details are below.

FUS Delta14 mice were generated by homologous recombination. Briefly, a construct of 8kb including a FRT-flanked neo cassette, was used to introduce an A to G splice mutation at g.13845 and "humanize" the coding sequence of exon 15 through the following sequence changes: g.14230 C>T, g.14232 A>T, g.14234C>G, g.14260 A>G and an ATTA insertion at g.14266. The construct was electroporated into in C57BL6/N-derived JM8-F6 embryonic stem (ES) cells. Correctly targeted clones were initially identified through long-range PCR and copy number qPCR and validated by RT-PCR and western blot for the truncated RNA/protein product, followed by DNA sequence verification. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred with B6-albino females (Stock No. 000058) for germline transmission. Offspring were bred with Flpo-expressing mice (Pgk1-FLPo on a C57BL/6 background; Stock No. 011065) for germline deletion of the PGK-neo-polyA cassette. The Neo-negative line then was backcrossed for at least three generations to C57BL/6J prior to sending males to The Jackson Laboratory in 2021. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Fus^tm1.1Emcf
Name: targeted mutation 1.1, Elizabeth MC Fisher
MGI accession ID: MGI:6100933
Generation method: Targeted
Attributes: Humanized sequence
Synonyms: FUS Delta14; Fus^tm1Emcf
Marker symbol: Fus
Marker name: fused in sarcoma
Marker synonyms: ALS6; altFUS; D430004D17Rik; D930039C12Rik; ETM4; FUS1; hnRNP P2; HNRNPP2; pigpen; POMP75; RGD1566093; Tls; translocated in liposarcoma
Chromosome: 7
Strain of origin: C57BL/6N
Molecular note: An A to G splice mutation was introduced betweeen exon 13 and 14 along with humanization of the coding sequence of exon 15 through the following sequence changes: g.14230 C to T, g.14232 A to T, g.14234 C to G, g.14260 A to G, and g.14266 ATTA insertion. Flp-mediated recombination removed the FRT-flanked neomycin resistance cassette.