STOCK Tg(CAG-dsRed2/RNAi:Tardbp)6Zxu/J

Stock number: 017934
Product name: amiR-TDP43i
Research areas: Cell Biology Research, Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

These amiR-TDP43i (or CAG-RFP-amiR-TDP43i) transgenic mice generally express a red fluorescent protein (dsRed2) and an artificial miRNA (amiR-TDP43) that targets the mouse TAR DNA binding protein mRNA for knockdown. Transgenic mice have reduced TDP-43 expression resulting in a rapid, progressive neurodegenerative phenotype that recapitulates many features of human amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

Detailed description

These amiR-TDP43i (or CAG-RFP-amiR-TDP43i) transgenic mice have widespread expression of both red fluorescent protein (dsRed2) and an artificial miRNA (amiR-TDP43) that targets the mouse TAR DNA binding protein mRNA for knockdown. Expression is directed by the CMV-IE enhancer/chicken β-actin/rabbit β-globin hybrid promoter. amiR-TDP43 expression in the central nervous system and periphery (including brain [both neurons and glia], spinal cord, muscle, liver and other organs) results in a modest reduction (~10-20%) of TDP-43 expression in central nervous system homogenates. dsRed2 expression may be observed by direct/native fluorescence, by mRNA (in situ) hybridization, and by antibody staining/immunohistochemistry. DsRed2 excitation and emission maxima are 563nm and 582nm, respectively. In the cortex, dsRed2 immunofluorescence is observed in ~30-40% of neurons, astrocytes and oligodendrocytes, but not in microglia. As expected, no GFP expression is reported in brain or spinal cord.

amiR–TDP43i hemizygotes are viable with no gross physical abnormalities at birth. Hemizygous mice may begin to display progressive neurological defects around the time they are weaned, including head shaking/bobbing, hyperactivity (~4-5 weeks), weight loss (~5 weeks), circling, wobbly gait (~6-7 weeks), hypoactivity (~10 weeks), paralysis (~9-20 weeks) and death by 9-20 weeks. amiR–TDP43i hemizygotes display three end-stage phenotypes: hindlimb paralysis, sudden death, and whole-body paralysis. Muscle denervation is evident by electromyography shortly after weaning. Progressive motor neuron degeneration results in the end disease stage, at which there is ~25% loss of layer 5 cortical neurons and ~60% loss of lumbar spinal cord ventral horn ChAT+ motor neurons. Many transgenic mice exhibit heart enlargement. The average lifespan is slightly shorter for males than females, but the difference is not statistically significant. The donating investigator has not attempted to generate homozygous mice to date (December 2012), and reports transgenic males breed better than transgenic females. Additionally, because the phenotype may become more severe if the mice are backcrossed with FVB/NJ for two or more generations, the colony is maintained at The Jackson Laboratory Repository by breeding transgenic males with (C57BL/6J x FVB/NJ)F1 hybrid females (Stock No. 019019) every generation.

Of note, the neuromuscular-phenotype-free, cre-inducible parental strain used to generate the amiR-TDP43i transgenic mice is also available at The Jackson Laboratory as Stock No. 017919 (amiR-TDP43u line 6 or CAG-loxp-EGFP/3xpA-loxp-RFP-amiR-TDP43).

Development

The CAG::loxP::EGFP/3xpA::loxP::dsRed2::intron::amiR-TDP43::intron::polyA transgene (or amiR-TDP43u transgene) was designed in the laboratory of Dr. Zuoshang Xu (University of Massachusetts Medical School). This transgene contained (from 5' to 3') the CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from the pCAGG vector), a loxP site, an enhanced green fluorescent protein sequence (from pEGFP-N1) followed by three polyadenylation signals, a second loxP site, a dsRed2 fluorescent protein sequence (from pDsRed2-C1), a synthetic artificial intron containing the amiR-TDP43 precursor miRNA sequence, and a polyA signal. The synthetic artificial intron utilized here was designed to contain typical intron splicing regulatory elements and was specifically placed 10 nucleotides downstream of the RFP stop codon because introns within 50 nucleotides downstream from the stop codon are found in some eukaryotic mRNAs and do not cause non-sense mediated decay (NMD). The amiR-TDP43 sequence (miR-TDP-43b; 5'-ATGCTGAACCTAAGCATAA-3') is an artificial precursor miRNA sequence encoding a short hairpin RNA specifically synthesized to target the RNA of mouse TAR DNA binding protein (TBP-43 or Tardbp). The resulting transgene was injected into (C57BL/6 x SJL)F2 hybrid mouse embryos. Transgenic mice from founder line 6 were identified, and bred with (C57BL/6J x FVB/NJ)F1 mice to establish germline transmission of the transgene. The amiR-TDP43u line 6 transgenic males (see Stock No. 017919) were then bred to mice with widespread expression of Cre recombinase (CMV-Cre on FVB genetic background) to remove the floxed EGFP sequences. The resulting amiR-TDP43i transgenic mice (line 6-50) were subsequently bred to (C57BL/6J x FVB/NJ)F1 female mice every generation for eight generations prior to sending to The Jackson Laboratory Repository. The donating investigator reports the CMV-Cre transgene was removed after two generations, and they selected breeders by high RFP fluorescence in tail samples. The donating investigator also reports amiR-TDP43i transgenic mice have two copies of the transgene inserted in head-to-head orientation on chromosome 14 band A2. Upon arrival at The Jackson Laboratory Repository, transgenic males were bred to (C57BL/6J x FVB/NJ)F1 hybrid females (Stock No. Stock No. 019019) every generation to maintain the colony.

Allele

Symbol: Tg(CAG-dsRed2/RNAi:Tardbp)6Zxu
Name: transgene insertion 6, Zuoshang Xu
MGI accession ID: MGI:5559582
Generation method: Transgenic
Attributes: Reporter; Knockdown
Marker symbol: Tg(CAG-dsRed2/RNAi:Tardbp)6Zxu
Marker name: transgene insertion 6, Zuoshang Xu
Chromosome: UN
Strain of origin: (C57BL/6 x SJL)F2
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: Widespread expression of both red fluorescent protein (dsRed2) and an artificial miRNA (amiR-TDP43). Expression of amiR-TDP43 in the central nervous system and periphery (including brain [both neurons and glia], spinal cord, muscle, liver and other organs) results in a modest reduction (~10-20%) of TDP-43 (Tardbp) expression in central nervous system homogenates.
Molecular note: The transgenic construct contains (from 5' to 3') the CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, a loxP site, an enhanced green fluorescent protein sequence followed by three polyadenylation signals, a second loxP site, a dsRed2 fluorescent protein sequence, a synthetic artificial intron containing the amiR-TDP43 precursor miRNA sequence, and a polyA signal. The synthetic artificial intron utilized here was designed to contain typical intron splicing regulatory elements and was specifically placed 10 nucleotides downstream of the RFP stop codon because introns within 50 nucleotides downstream from the stop codon are found in some eukaryotic mRNAs and do not cause non-sense mediated decay (NMD). The amiR-TDP43 sequence (miR-TDP-43b; 5'-ATGCTGAACCTAAGCATAA-3') is an artificial precursor miRNA sequence encoding a short hairpin RNA specifically synthesized to target the RNA of mouse TAR DNA binding protein (TBP-43 or Tardbp).