These knock-in mice contain a floxed neomycin cassette (neoR) in opposite orientation to a mutated Nlrp3 gene resembling the human mutation associated with Muckle-Wells syndrome. When bred to mice that express Cre recombinase to delete the floxed-neoR, the mutant gene is expressed in cre-expressing tissues of the offspring. These mice may be useful in studying the role of cryopyrin in the regulation of autoinflammatory diseases.
Hal M Hoffman, UCSD
Nlrp3A350VneoR mice contain a floxed neomycin cassette in intron 2 of the NLR family, pyrin domain containing 3 gene, Nlrp3, in opposite orientation to gene (neoR). These mice also contain a point mutation in exon 3 which results in a missense mutation, A350V, corresponding to human amino acid 352. This mutation is commonly found in humans with Muckle-Wells syndrome (MWS). NLRP3 encodes the protein cryopyrin, which is a cytosolic nucleotide-binding domain and leucine-rich repeat containing (NLR) protein expressed in white blood cells and chondrocytes. Cryopyrin controls the formation of the inflammasome which regulates the immune system's response to injury, toxins, and infection by cleaving interleukin (IL)-1β. NLRP3 mutations are known to cause autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS) such as MWS, familial cold autoinflammatory syndrome (FCAS), and neonatal onset multisystem inflammatory disease (NOMID). Homozygotes are viable and fertile. When bred to mice that express Cre recombinase, resulting offspring will have the floxed-neoR deleted in the cre-expressing tissues, allowing expression of the mutant gene.
For example, when bred to B6.129P2-Lyz2tm1(cre)Ifo/J mice (Stock No. 004781), Nlrp3A350VneoR expression in myeloid lineage cells results in postnatal lethality, with 70% of mice dying by 14 days of age. Neutrophilic infiltrates are evident in skin, liver, spleen, joint, sinus, conjunctiva, bone marrow, and tongue. They lack hair and have red scaly skin, and exhibit extreme necrosis of the gut and kidney.
When bred to mice carrying Tg(CAG-(cre/Esr1*)5Amc (Stock No. 004682), tamoxifen-induced Cre-mediated recombination results in hypersecretion of IL1b and an enhanced ability to stimulation T cell differentiation by activated dendritic cells.
When bred to mice carrying Tg(Zp3-cre)3Mrt (Stock No. 003394), Cre recombinase expression in the oocyte results in postnatal lethality from multi-organ failure following inflammation and necrosis.
A targeting vector was designed to insert a loxP-flanked neomycin resistance (neo) cassette, in reverse orientation to the gene, into intron 2 of the NLR family, pyrin domain containing 3 gene, Nlrp3. A point mutation was introduced into exon 3,resulting in a missense mutation, A350V, corresponding to human amino acid 352. This mutation is commonly found in humans with cryopyrin-associated periodic syndromes (CAPS). The construct was electroporated into 129/SvJ-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and chimeric mice were bred to C57BL/6NCrl mice. The donating investigator reports that mutant mice were backcrossed to C57BL/6NCrl for at least ten generations (see SNP note below) prior to sending to The Jackson Laboratory. Upon arrival, heterozygous sperm was frozen. To generate the live colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6J genetic background.
|Allele Name||targeted mutation 1, Hal M Hoffman|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Humanized sequence, No functional change)|
|Gene Symbol and Name||Nlrp3, NLR family, pyrin domain containing 3|
|Promoter||Nlrp3, NLR family, pyrin domain containing 3, mouse, laboratory|
|Strain of Origin||129/Sv|
|Molecular Note||The alanine 352 to valine human mutation associated with Muckle-Wells syndrome was produced in mice by changing the equivalent alanine at position 350 to a valine (A350V). A floxed neomycin cassette was inserted in the reverse orientation into intron 2, which is upstream of the point mutation in exon 3. Sequence analysis of transcripts isolated from heterozygote mice demonstrate the mutant allele is not expressed due to the presence of the neomycin cassette. In the presence of cre recombinase, the neomycin cassette is removed and the mutant allele is expressed.|
|Mutations Made By|| |
Hal Hoffman, UCSD
When maintaining a live colony, homozygous mice may be bred together.
When using the Nlrp3A350VneoR mouse strain in a publication, please cite the originating article(s) and include JAX stock #017969 in your Materials and Methods section.