B6.129S4-Gt(ROSA)26Sor^{tm1(CAG-C9orf72)Lgu}/J

Stock number: 032108
Product name: R26-CAG-LSL-C9orf72 , Cre-inducible C9orf72 overexpression
Research areas: Neurobiology Research, Research Tools

Description

R26-CAG-LSL-C9orf72 mice allow Cre recombinase-inducible C9ORF72 overexpression. These mice may be useful for studying amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) and frontotemporal dementia (FTD).

Detailed description

Mutations in the C9orf72 member of C9orf72-SMCR8 complex gene (C9orf72) are associated with human amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

R26-CAG-LSL-C9orf72 is a Gt(ROSA)26Sor knock-in allele with a CAG promoter and loxP-flanked STOP cassette upstream of a mouse C9orf72 cDNA sequence. These R26-CAG-LSL-C9orf72 mice allow Cre recombinase-inducible C9ORF72 overexpression.

Prior to Cre recombinase exposure, the STOP cassette is designed to prevent transcription of the downstream C9orf72. The donating investigator reports R26-CAG-LSL-C9orf72 mice exhibit C9ORF72 qPCR levels that are similar to wildtype (see note on western blot below). Mice heterozygous or homozygous for the R26-CAG-LSL-C9orf72 allele are viable and fertile with normal breeding, and no reported gross physical or behavioral abnormalities.

Following Cre recombinase exposure, deletion of the STOP cassette in the cre-expressing tissues allows the downstream C9orf72 to be expressed (as well as retaining normal expression of the endogenous C9orf72 locus). The resulting R26-CAG-C9orf72 allele is therefore predicted to have total C9ORF72 overexpression. Specifically, when R26-CAG-LSL-C9orf72 are bred to germline Cre deleter mice (Stra8-iCre; see Stock No. 017490), the donating investigator reports the resulting homozygous C9ORF72 overexpression mice exhibit significantly increased C9ORF72 qPCR levels (see note on western blot below), as well as a metabolic phenotype that is currently being characterized (November 2018).

Of note, the donating investigator reports several approaches for western blot analyses have been performed to date (November 2018), but the results are confounded due to difficulties with commercially-available C9orf72 antibodies.

Also of note, the R26-CAG-LSL-C9orf72 allele has frt sites flanking the loxP-STOP-loxP::C9orf72::WPRE::polyA sequences. This allows for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired.

Development

The R26-CAG-LSL-C9orf72 allele was created in the laboratory of Dr. Leonard P. Guarente (Massachusetts Institute of Technology) to have a CAG promoter and loxP-flanked STOP cassette upstream of a mouse C9orf72 gene, all inserted into the Gt(ROSA)26Sor locus. The specific details are below.

The donating investigator used a targeting vector designed to have (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a frt site, a loxP site, a transcriptional/translational STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a second loxP site, a cDNA sequence encoding the mouse C9orf72 member of C9orf72-SMCR8 complex (C9orf72), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, a second frt site and a neo cassette.

The donating investigator reports the CAG::frt::loxP-STOP-loxP::C9orf72::WPRE::polyA::frt::neo construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into (C57BL/6 x 129S4/SvJae)F1-derived v6.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric animals were bred to C57BL/6 mice for germline transmission of the R26-CAG-LSL-C9orf72 allele. The colony was maintained by breeding R26-CAG-LSL-C9orf72 mice together and/or to C57BL/6J inbred mice for 3-4 generations. In 2018, R26-CAG-LSL-C9orf72 males (black coat color) were sent to The Jackson Laboratory Repository. Upon arrival, they were bred at least one generation to C57BL/6J inbred mice (Stock No. 000664). The resulting R26-CAG-LSL-C9orf72 colony was analyzed by 148 SNP panel analysis and determined to be C57BL/6J congenic (~98% C57BL/6J). This mouse line was then cryopreserved in 2019.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-C9orf72)Lgu}
Name: targeted mutation 1, Leonard Guarente
MGI accession ID: MGI:6241353
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence
Synonyms: R26-CAG-LSL-C9orf72
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: The targeting vector is designed to have (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP site, a transcriptional/translational STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a second loxP site, a cDNA sequence encoding the mouse C9orf72 member of C9orf72-SMCR8 complex (C9orf72), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a BGH polyA signal, a second FRT site and a neo cassette. The construct is inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.