These MRL/lpr NOS3-/- mice carry the Nos3tm1Unc targeted mutation and Faslpr spontaneous alleles on the MRL genetic background, and may be useful in studies of autoimmune diseases such as systemic lupus erythematosus (SLE).
James C. Oates, Medical University of South Carolina
Mice that are homozygous for both the targeted mutation and spontaneous mutant alleles on this genetic background are viable, but die prematurely. Female mice that are homozygous for both mutations have reduced fertility. MRL.Cg-Nos3tm1Unc Faslpr/J double homozygotes die as early as 6 weeks of age, as compared to an average of 17 weeks of age lifespan for female MRL/MpJ-Faslpr/J mice and 22 weeks of age for MRL/MpJ-Faslpr/J males. MRL/lpr NOS3-/- mice exhibit increased proteinuria and urine albumin levels, decreased urine total nitrate and nitrite content and more severe proliferative
lupus nephritis (greater hypercellularity, necrosis, crescent formation, chronic inflammation, and vasculitis) when compared to MRL/MpJ-Faslpr/J wildtype animals. CD4+ T cells from double homozygotes have diminished nitric oxide production and mitochondrial calcium levels. Superoxide production in renal cortical tissue from MRL.Cg-Nos3tm1Unc Faslpr/J homozygotes is increased compared to MRL.Cg-Nos2tm1Lau Faslpr/J homozygotes (Stock No. 022350).
Mice carrying the Nos3tm1Unc allele (on a mixed C57BL/6 and 129P2/OlaHsd background) were obtained by the Donating Investigator from JAX (Stock No. 002684). Briefly, a targeting vector designed by Dr. Oliver Smithies (University of North Carolina) and containing neomycin resistance and herpes simplex virus thymidine kinase genes was used to replace 129 bp of exon 12, which disrupted the calmodulin binding domain. The construct was electroporated into 129P2/OlaHsd-derived E14TG2a embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts and the resulting chimeric males were crossed to C57BL/6 female mice. These B6;129P2-Nos3tm1Unc mice were crossed to MRL/MpJ-Faslpr/J (JAX Stock No. 000485) mice for 9 generations using a marker assisted protocol. Upon arrival at The Jackson Laboratory, the mice were crossed to MRL/MpJ-Faslpr/J (Stock No. 000485) at least once to establish the colony.
|Allele Name||targeted mutation 1, University of North Carolina|
|Allele Type||Targeted (Null/Knockout)|
|Allele Synonym(s)||ecNOS-; eNOS-; eNOSKO; NOS3-|
|Gene Symbol and Name||Nos3, nitric oxide synthase 3, endothelial cell|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||A 1.2 kb neomycin cassette replaced 129 bp of exon 12 of the gene. This disrupted the calmodulin binding site of the protein and introduced a premature stop codon into the transcripts. Immunohistochemisty of heart and kidney sections from homozygous mutant mice confirmed that no detectable encoded protein was present.|
|Mutations Made By|| |
Dr. Oliver Smithies, University of North Carolina at Chapel Hill
|Allele Type||Spontaneous (Hypomorph)|
|Allele Synonym(s)||Fas-; Fas-def; lpr; MRL/lpr; Tnfrf6lpr; Tnfrsf6lpr; Tnfrsf6lpr|
|Gene Symbol and Name||Fas, Fas (TNF receptor superfamily member 6)|
|Strain of Origin||MRL/Mp|
|General Note||Faslpr, lymphoproliferation, recessive. This mutation was found during inbreeding of a strain MRL/Mp derived from crosses among strains LG, AKR, C3H, and C57BL/6. The resemblance has led to extensive use of Faslpr mice in attempts to determine the etiology of SLE and to evaluate therapies. However, the human APT1 gene (OMIM 134637) encodes the FAS antigen; Tnfrsf6 is not the homolog of the human (SLE) gene.The Cd72c haplotype is a modifier of Faslpr-induced autoimmune disease. J:204782|
|Molecular Note||Southern blotting experiments indicated that the mutation is a genomic rearrangement within the gene, probably within the second intron. Sequencing of genomic DNA and RT-PCR products from homozygous mutant mice revealed the insertion of an early transposable element (ETn) into intron 2. RT-PCR analysis of liver and thymus mRNA showed that the presence of the ETn leads to premature termination of transcription at the long terminal repeat (LTR) of the ETn and aberrant mRNA splicing. The mutation is "leaky," however, as full-length mRNA and a longer splice product incorporating a segment of the ETn as an extra intron are detected in the thymus at low levels.|
When maintaining a live colony, these mice that are heterozygous for the Nos3tm1Unc allele and homozygous for the Faslpr allele can be bred together. Mice that are homozygous for both alleles have reduced survival and can die as early as 6 weeks of age. Female mice that are homozygous for both mutations have reduced fertility.
When using the MRL.Cg-Nos3tm1Unc Faslpr/J mouse strain in a publication, please cite the originating article(s) and include JAX stock #022760 in your Materials and Methods section.
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