C57BL/6J-Tg(CAG-lacZ,-FUS*R521G,-EGFP)673Gyu/J

Stock number: 028021
Product name: CAG-Z-FUS^R521G-IRES-EGFP transgenic line 673 [CAG-Z-FUS^R521G (673)]
Research areas: Cancer Research, Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

The CAG-Z-FUS^R521G-IRES-EGFP transgenic line 673 [or CAG-Z-FUS^R521G (673)] has widespread expression of lacZ, that is replaced by expression of the human fused in sarcoma R521G mutant isoform (huFUS^R521G) and EGFP fluorescence following exposure to Cre recombinase. The Cre recombinase-inducible expression of huFUS^R521G, along with similarly designed transgenic mice harboring the human wildtype FUS (Stock No. 027898), is useful in studying FUS-linked juvenile amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease), and the neurodegenerative disorder frontotemporal lobar degeneration (FTLD).

Detailed description

Nucleo-cytoplasmic shuttling of fused in sarcoma (FUS) occurs by a nuclear localization signal (NLS). The majority of clinical amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) and frontotemporal lobar degeneration (FTLD)-associated FUS mutations occur in its C-terminal NLS sequence. Overexpression of wildtype FUS protein can be pathogenic in human patients as well.

CAG-Z-FUS^R521G-IRES-EGFP transgenic line 673 (simply called CAG-Z-FUS^R521G (673)) has a floxed-lacZ gene preventing transcription of the downstream bicistronic sequences; a human fused in sarcoma R521G mutant isoform (huFUS^R521G) and EGFP. Prior to Cre recombinase exposure, lacZ expression is directed to widespread tissues by the CAG promoter, and no expression of huFUS^R521G or EGFP is observed. When bred to mice that express Cre recombinase, the resulting offspring will have the floxed-lacZ deleted in the cre-expressing tissues; resulting in expression of huFUS^R521G and EGFP fluorescence in cre-expressing tissues (and continued lacZ expression in other tissues).

Mice hemizygous for the CAG-Z-FUS^R521G transgene are viable and fertile, born at normal Mendelian ratios, and have no reported phenotypic abnormalities. To date (September 2015), it has not been attempted to make this strain homozygous.

When CAG-Z-FUS^R521G (673) are bred to C57BL/6-congenic Meox2^Cre knock-in animals (Stock No. 003755), germline deletion of floxed-lacZ generates the CAG-FUS^R521G-IRES-EGFP genotype (simply called CAG-FUS^R521G). Mice hemizygous for the CAG-FUS^R521G transgene and heterozygous for Meox2^Cre (Tg/+ , +/Cre) have global overexpression of low levels of huFUS^WT, resulting in severe deficits in motor function, denervation of the neuromuscular junctions (NMJs), muscle atrophy, neuroinflammation and early lethality (~70% die by postnatal day 30). The level of huFUS^R521G expression was similar to that of endogenous mouse FUS, resulting in ~2-fold expression levels of total FUS (huFUS^R521G + mouse FUS) compared to control mice. CAG-FUS^R521G mice that survive early lethality have impairments in motor function and sociability. Although FUS^R521G is not overtly mislocalized in these animals (presumably due to having low levels of overexpression rather than high levels of overexpression), it is not yet determined if the shuttling dynamics of FUS^R521G are altered.

In addition, both CAG-FUS^WT (Stock No. 027898 with germline deletion of floxed-lacZ) and CAG-FUS^R521G transgenic mouse models exhibit no overt motor neuron loss, no apparent ubiquitin-positive aggregation and no mislocalization of FUS in neurons and glia. Transcriptomic analysis of spinal cords revealed that the gene expression pattern is altered in CAG-FUS^WT mice, but not significantly altered in CAG-FUS^R521G mice. The CAG-FUS^WT and CAG-FUS^R521G animals display some characteristics of adult ALS. However, the onset of phenotype in the mouse models is earlier, more closely reflecting FUS-linked juvenile ALS.

Development

The CAG-Z-FUS^R521G-IRES-EGFP transgene was created by Dr. Gang Yu (University of Texas Southwestern Medical Center at Dallas). The transgene was designed with (from 5' to 3') the CMV-IE enhancer/chicken beta-actin hybrid promoter (CAG), a loxP site, the β-galactosidase gene (lacZ) with nuclear translocation signal and triple polyadenylation sequence, a second loxP site, a cDNA sequence encoding the human fused in sarcoma R521G mutant isoform (huFUS^R521G; encoding an autosomal recessive missense mutation in the nuclear localization signal at the C-terminus of FUS that is associated with amyotrophic lateral sclerosis), an internal ribosome entry site sequence (IRES), the enhanced green fluorescent protein gene (EGFP) and a polyadenylation sequence. The ~13.2 kbp construct was injected into fertilized oocytes from C57BL/6J female mice. Transgenic founders were bred to C57BL/6J animals for germline transmission. CAG-Z-FUS^R521G-IRES-EGFP founder line 673 (simply called CAG-Z-FUS^R521G (673)) was identified with single-to-very low copies of the transgene at a single insertion site. CAG-Z-FUS^R521G (673) was backcrossed to C57BL/6J for at least 13 generations prior to sending to The Jackson Laboratory Repository in 2016. Upon arrival, males were used to cryopreserve sperm. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

 

Allele

Symbol: Tg(CAG-lacZ,-FUS*R521G,-EGFP)673Gyu
Name: transgene insertion 673, Gang Yu
MGI accession ID: MGI:5662097
Generation method: Transgenic
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(CAG-lacZ,-FUS*R521G,-EGFP)673Gyu
Marker name: transgene insertion 673, Gang Yu
Chromosome: UN
Strain of origin: C57BL/6J
Expressed genes: FUS,fused in sarcoma ,human; GFP,Green Fluorescent Protein,; lacZ,beta-galactosidase,E. coli
Site of expression: Prior to cre recombination lacZ is widely expressed; the human fused in sarcoma R521G mutant isoform and EGFP are expressed only after cre recombination in cre-expressing tissues.
Molecular note: This transgene consists of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter, a floxed lacZ gene, human FUS cDNA with the R521C mutation, IRES, and EGFP coding sequences. Cre-excision of the loxP flanked lacZ DNA allows for translation of FUS and EGFP. Two lines were generated: 673, and 682. Line 682 was chosen as a representative line.