B6.Cg-S1pr1^tm1.1Thla/J

Stock number: 030439
Product name: S1pr1^S5A
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

This S1pr1^S5A strain produces a phosphorylation and internalization deficient protein and exhibits a more severe experimental autoimmune encephalomyelitis (EAE) as compared to controls. These mice may be suitable for use in studies related to lymphocyte egress from lymphoid organs, leukocyte migration, and autoimmunity.

Detailed description

The targeted S1pr1 gene encodes a receptor for sphingosine 1-phosphate that is highly expressed in endothelial cells, and is involved in regulation of cell adhesion, cell migration, angiogenesis, and lymphocyte trafficking. These mice carry a mutant allele of S1pr1 that produces an internalization-deficient protein in which the C-terminal serine cluster has been substituted with alanines (nucleotide substitutions in exon 2 that result in amino acid substitutions S351A, S353A, S355A, S358A, and S359A). The point mutations in exon 2 were confirmed by sequencing. G-protein dependent signaling is not disrupted in mutant mice. Homozygotes exhibit increased splenic CD4+ and CD8+ T cell cell numbers, delayed inhibitor-induced (FTY720) lymphopenia, and more severe experimental autoimmune encephalomyelitis (EAE) compared to controls. Pulmonary vascular leakage and receptor degradation due to S1P1 modulator treatment is reduced in mutant mice. Homozygotes are viable and fertile. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.

Development

A targeting vector containing loxP site flanked neo cassette and nucleotide substitutions in exon 2 that result in amino acid substitutions S351A, S353A, S355A, S358A, and S359A, was electroporated into unspecified 129S6 × C57BL/6 F1 derived embryonic stem (ES) cells (University of Connecticut Health Center). Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission, and were crossed to mice expressing FLP1 recombinase on the 129S4/SvJaeSor genetic background (Stock No. 003946) to excise the FRT site flanked neo cassette, and were then backcrossed to C57BL/6J for 5 generations. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background. The mice no longer contain the FLP recombinase allele. Sequencing confirmed the point mutations in exon 2. 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).

Allele

Symbol: S1pr1^tm1.1Thla
Name: targeted mutation 1.1, Timothy Hla
MGI accession ID: MGI:4822471
Generation method: Targeted
Synonyms: S1p1r^S5A
Marker symbol: S1pr1
Marker name: sphingosine-1-phosphate receptor 1
Marker synonyms: CD363; CHEDG1; D1S3362; ECGF1; Edg1; EDG-1; S1P; S1P1
Chromosome: 3
Strain of origin: (129S6/SvEvTac x C57BL/6)F1
Molecular note: Nucleotide substitutions in exon 2 result in the amino acid substitution of five serines with alanines (p.[Ser351Ala;Ser353Ala;Ser355Ala] and p.(Ser358_Ser359delinsAlaAla)). This prevents phosphorylization of the five residues and the produced protein fails to internalize correctly, but signaling is not disrupted. A loxP site flanked neomycin resistance gene cassette inserted into intron 2 was removed by subsequent cre-mediated recombination.