B6;129S6-Gt(ROSA)26Sor^{tm1(TARDBP*M337V/Ypet)Tlbt}/J

Stock number: 029266
Product name: TDP-43^M337V BAC knock-in
Research areas: Neurobiology Research, Research Tools

Description

TDP-43^M337V BAC allele is a Gt(ROSA)26Sor knock-in of a loxP-flanked, full-length human TARDBP genomic locus with M337V mutation in exon 6 and C-terminal fluorescent tag. Prior to Cre recombinase exposure, TDP-43^M337V BAC mice have stable, single-copy, low-level expression of TDP-43^M337V-Ypet that recapitulates many of the key features of ALS/FTD (amyotrophic lateral sclerosis [Lou Gehrig's disease] / frontotemporal dementia [frontotemporal lobar degeneration]), including progressive motor deficits in conjunction with neuromuscular junction abnormalities and reduced survival.

Detailed description

The human TAR DNA binding protein (TARDBP or TDP-43) is an ubiquinated, DNA/RNA-binding protein localized to the nucleus of cells. TDP-43 mutations in the amyloidogenic core region (residues 311-360) and the glycine-rich, flexible C-terminal region are associated with the development of amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) and frontotemporal dementia (FTD or frontotemporal lobar degeneration). The TDP-43 methionine-to-valine (M337V) missense mutation is associated with increased low molecular weight aggregates and neuronal dysfunction.

The TDP-43^M337V BAC allele has a loxP-flanked, full-length human TARDBP genomic locus with M337V mutation in exon 6 and C-terminal fluorescent tag, all inserted into the Gt(ROSA)26Sor locus. Exposure to Cre recombinase leads to deletion of the TDP-43^M337V BAC sequences. The phenotype of TDP-43^M337V BAC mice prior to Cre recombinase is described below.

In contrast to transgene overexpression models, this Gt(ROSA)26Sor knock-in TDP-43^M337V BAC allele exhibits stable, single-copy, low-level expression of TDP-43^M337V-Ypet driven by the endogenous human promoter. Both homozygous and heterozygous mice of both sexes recapitulate many of the key features of ALS/FTD; including progressive motor deficits in conjunction with neuromuscular junction (NMJ) abnormalities and reduced survival.


At 12 months of age, despite a clear neuromuscular phenotype, homozygotes and heterozygotes do not demonstrate cytoplasmic mislocalization/accumulation of TDP-43 - the key pathological hallmark of ALS in the CNS.

Reduced survival is observed in both sexes (homozygotes ~500 days; heterozygotes ~650 days). The phenotype is dose-dependent: homozygous mice have an earlier age-of-onset and are more severely affected than heterozygous littermates. In addition, cultured spinal cord motor neurons derived from heterozygous embryos show a characteristic phenotype of reduced survival, translocation of TDP-43 from nucleus to cytoplasm and reduced stress granule formation.
Homozygous and heterozygous mice are viable and fertile, with no apparent deficits in fertility or litter size compared to controls. Human TDP-43^M337V-Ypet is expressed at low levels in all tissues examined in the expected pattern of cellular distribution, with no significant expression level differences reported between heterozygous or homozygous mice (cortex and spinal cord). In heterozygous mice, the TDP-43^M337V-Ypet expression level is ~40% of endogenous levels.
The donating investigator reports that C-terminal Ypet tag is suitable for immunostaining via antibody directed against GFP, but not for direct fluorescence.

Development

The TDP-43^M337V BAC mice were created by collaboration led by Dr. Kevin Talbot (University of Oxford). BAC cloning was performed in the laboratory of Dr. Richard Wade-Martins (University of Oxford), using the RP11-829B14 BAC as source DNA for the full-length human TARDBP genomic locus. The human TARDBP was then modified by site-directed mutagenesis to create the M337V missense mutation in exon 6 (an A-to-G base change [A1009G] encoding a methionine-to-valine substitution at amino acid position 337). The final TDP-43-M337V-Ypet BAC construct includes the TDP-43-M337V genomic DNA (44,886 bp) with a Ypet ORF cDNA sequence (717 bp; described below) fused to the 5' end of TARDBP exon 6, as well as 9.2 kbp of the original BAC backbone sequences. The TDP-43-M337V-Ypet BAC sequences are flanked with loxP sites. In the mouse core facility directed by Dr. Ben Davies (Wellcome Trust Centre for Human Genetics, University of Oxford), (C57BL/6J x 129S6/SvEvTac)F1-derived IDG26.10-3 embryonic stem (ES) cells with a pre-integrated Gt(ROSA)26Sor locus (splice acceptor::frt::PGK promoter-attP-hygromycin resistance gene-pA::frt::attP between exons 1 and 2) were re-transfected with the TDP-43-M337V-Ypet BAC sequence and a PhiC31 vector for integrase-mediated cassette exchange. The correctly re-targeted ES cells, with the Gt(ROSA)26Sor containing (from 5' to 3') splice acceptor::frt::PGK promoter-attR-neomycin resistance gene-bGHpA::loxP::TDP-43-M337V-Ypet BAC sequence::loxP::attL, were injected into recipient blastocysts. Chimeric males were bred with C57BL/6J females to establish germline transmission. The resulting TDP-43^M337V BAC colony was additionally backcrossed to C57BL/6J wildtype mice for three generations prior to sending homozygous males with black coat color to The Jackson Laboratory Repository in 2016. 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).
Of note, the donating investigator reports that the Y chromosome may not have been fixed to the C57BL/6J background during backcrossing.
The Ypet cDNA sequence used here is described in Nguyen and Daugherty 2005 Nat Biotechnol. 23:355. The codon-improved YFP variant YFP3 (with F46L, I47L, S208F, V224L, H231E, D234N mutations) was further modified to include several Venus mutations (F64L, R79K, M153T, V163A, S175G), resulting in the YFP for energy transfer (YPet) variant.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(TARDBP*M337V/Ypet)Tlbt}
Name: targeted mutation 1, Kevin Talbot
MGI accession ID: MGI:5784728
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence; Humanized sequence
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6J x 129S6/SvEvTac)F1
Expressed genes: TARDBP,TAR DNA binding protein,human; YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: When these mutant mice are bred to mice that express Cre recombinase, the resulting offspring will express YFP (revealed by immunostaining via antibody directed against GFP) in the cre-expressing tissues.
Molecular note: The human TARDBP from BAC RP11-829B14 was modified by site-directed mutagenesis to create the M337V missense mutation in exon 6 (an A-to-G base change (A1009G) encoding a methionine-to-valine substitution at amino acid position 337). The final construct includes the TDP-43-M337V genomic DNA (44,886 bp) with a Ypet ORF cDNA sequence (717 bp; described below) fused to the 5' end of TARDBP exon 6, as well as 9.2 kbp of the original BAC backbone sequences. The TDP-43-M337V-Ypet BAC sequences are flanked with loxP sites. Embryonic stem (ES) cells with a pre-integrated Gt(ROSA)26Sor locus (splice acceptor::FRT::PGK promoter-attP-hygromycin resistance gene-pA::FRT::attP between exons 1 and 2) were re-transfected with the TDP-43-M337V-Ypet BAC sequence and a PhiC31 vector for integrase-mediated cassette exchange. The Ypet cDNA sequence is a codon-improved YFP variant YFP3 (with F46L, I47L, S208F, V224L, H231E, D234N mutations) further modified to include several Venus mutations (F64L, R79K, M153T, V163A, S175G), resulting in the YFP for energy transfer (YPet) variant.
General note: embryonic stem cell line = IDG26.10-3