The most common genetic mutation associated with human amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in a non-coding region of the chromosome 9 open reading frame 72 gene (C9orf72). The mouse 3110043O21Rik locus is the orthologue of human C9orf72.

The C9orf72 BAC transgenic line C9-500 expresses a full-length human C9orf72 gene with ~500 hexanucleotide repeat expansions (GGGGCC; or G4C2) in intron 1a. The BAC includes large regions (~52 kbp upstream and ~19 kbp downstream) flanking the complete C9orf72 gene but no other complete annotated genes. The donating investigator indicates that the large amount of flanking sequences are likely to contain regulatory regions needed to control sense/antisense transcript expression in a temporal and spatial pattern that mimics expression in the human disease. The hexanucleotide repeat size is generally stable but should be monitored (see further discussion below). The transgene copy number and expression levels are also described in greater detail below.

Please note: The Jackson Laboratory has received inquiries noting that this strain was not demonstrating some of the initial phenotypes reported in the original publication (Liu et al. 2016 Neuron 90:521). In particular that the mice did not develop paralysis. In an independent investigation at The Jackson Laboratory involving over 100 hemizygous females and controls aged more than 15 months, we noted that the mice exhibit some of the key features noted in the publication, including hyperactivity, kyphosis and lethality in a small percentage of mice. However, in our hands, these phenotypes were seen in the same frequency as wildtype (noncarrier) control animals. The nature of the lethalities also appear to correlate with seizures, a trait noted in the FVB/N background. No indication of paralysis were observed in the mice on the study performed at The Jackson Laboratory, and Compound Muscle Action Potential (CMAP) did not vary between hemizygous animals and controls. It should be noted that in the original publication, the penetrance of this phenotype was low. Considerations such as gene by environment interactions can play a part in phenotypic manifestations, and we continue to work closely with the donating investigator and the scientific community to resolve these features in this mouse model. The Jackson Laboratory has confirmed that mice do express the transgene and that dipeptide repeat (DPR) levels of polyGP (as measured by ELISA) are 100X higher than background at two months of age. [2019]

The phenotype of hemizygous C9-500 mice as published in Liu et al. 2016 Neuron 90:521 is described in detail below.
Hemizygous C9-500 mice are a model of C9orf72 ALS/FTD that shows decreased survival, paralysis, muscle denervation, motor neuron loss, anxiety-like behavior, and cortical and hippocampal neurodegeneration. These mice express C9orf72 sense transcripts and upregulated antisense transcripts. In contrast to sense RNA foci, antisense foci preferentially accumulate in ALS/FTD-vulnerable cell populations. RAN protein accumulation increases with age and disease, and TDP-43 inclusions are found in degenerating brain regions in end-stage animals. C9-500 mice exhibit both an acute, rapidly progressive disease as well as a slow progressive disease.
Hemizygous C9-500 animals appear normal with no overt cage behavior abnormalities up to 16 weeks of age. Between 20-40 weeks of age, ~30-35% of C9-500 females develop an acute, rapidly progressive disease characterized by inactivity, labored breathing, sudden weight loss, hindlimb weakness, motor neuron degeneration/disease throughout the motor unit, paralysis and death. Acute mice exhibit molecular hallmark features of C9orf72 ALS/FTD, including upregulation of C9orf72 antisense transcripts at high levels in the frontal cortex (similar to autopsy tissue from C9(+) ALS patients), and to a lesser extent in the spinal cord. Acute end-stage mice have widespread neurodegenerative changes in the neocortex, hippocampus, cerebellum and spinal cord. Specifically, poly(GA) RAN protein aggregation is abundant, TDP-43 inclusions are found in degenerating brain regions, and widespread pyknotic nuclei, vacuolization and gliosis are evident. Hemizygous C9-500 males do not exhibit an acute phenotype.
Hemizygous C9-500 animals may also exhibit a slow progressive disease. At 1 year of age, ~40-46% of C9-500 females have a milder and slower progressive disease characterized by kyphosis, reduced activity, hyperactivity when provoked, clasping and intermittent seizures. ~43%-45% of C9-500 males develop symptoms similar to the slow progressive female mice. Compared to the acute disease animals, the slow progressive mice show subtle motor neuron degeneration/disease characteristics. They exhibit loss of motor neurons in the spinal cord, focal sites of neurodegeneration in the neocortex, and milder degeneration of the cerebellum with relative sparing of the hippocampus.

Hemizygous mice are viable and fertile with the phenotype described above. To date (April 2019), the phenotype of homozygous mice has not been characterized.

Regarding hexanucleotide repeat size/stability, the donating investigator reports that C9-500 mice show a single hexanucleotide repeat size (~500) that is relatively stable between generations in the large majority of mice. When using mouse models where repeat number is subject to germline and somatic instability, and may expand or contract, it is strongly recommended the repeat number be quantified in all the experimental animals; all animals in all experimental groups should carry comparable hexanucleotide repeat sizes.

Regarding transgene copy number and expression levels, hemizygous C9-500 mice show overall levels of C9orf72 sense transcripts that correlate to that expected for a single transgene copy, and the expression levels of C9ORF72_[500] and 3110043O21Rik (each relative to beta-actin) are comparable. In August 2019, The Jackson Laboratory performed additional transgene insertion analyses on hemizygous mice. This suggested a single copy of the transgene has integrated on chromosome 6 (114,939,853-114,939,873 [mouse mm10]) and resulted in a 20 bp deletion of genomic region in between the identified breakpoints. There are currently no genes annotated in this region. No other genomic rearrangements are evident.

The Jackson Laboratory Southern Blot Protocol for this transgene is available here [2019]. Using this protocol, the expected band size for Stock No. 029099 is ~6 kbp. Anything above is ruled as an expansion, anything below is ruled a contraction.