B6.Cg-S1pr1^tm4.1Rlp Gt(ROSA)26Sor^{tm2.1(luc*/Arrb2*)Rlp} Tyr^c-2J/J

Stock number: 033605
Product name: S1P₁ luciferase signaling mice
Research areas: Dermatology Research, Mouse/Human Gene Homologs, Research Tools, Sensorineural Research

Description

S1P₁ luciferase signaling mice enable the bioluminescent detection of G protein-coupled receptor (GPCR) activation in real time.

Detailed description

S1P₁ luciferase signaling mice have a C-terminal luciferase fragment, with a polyadenylation signal, inserted into the C-terminus of the endogenous sphingosine-1-phosphate receptor 1 (S1pr1) gene. They also contain an N-terminal luciferase fragment inserted into the N-terminus of the β-arrestin2 (Arrb2) gene, integrated into the Gt(ROSA)26Sor locus.

S1P₁ has a high affinity for the bioactive sphingosine-1-phosphate (S1P) and is highly enriched in endothelial cells, where it serves as a key regulator of vascular barrier function. S1P₁ also performs important actions in the immune and nervous systems. S1P- S1P₁ signaling has been linked to disease processes including infection, multiple sclerosis, atherosclerosis, inflammatory bowel disease, and cancer. β-arrestin binding to activated G protein-coupled receptors (GPCRs) also mediates receptor desensitization, internalization, and recycling.

This strain enables the bioluminescent detection of GPCR activation in real time. Upon receptor activation and subsequent β-arrestin2 recruitment, an active luciferase enzyme complex is produced, which can be detected by in vivo bioluminescence imaging. This imaging strategy reveals the dynamics and spatial specificity of S1P₁ activation in normal and pathophysiologic contexts in vivo. Mice that are heterozygous for the S1P₁-CLuc allele and homozygous for the NLuc-β-arrestin2 were studied due to higher bioluminescence signal after activation of S1P₁ than mice heterozygous for both of these alleles. Mice homozygous for both alleles are viable and fertile.

Development

The S1P₁-CLuc targeting construct was designed to insert a C-terminal luciferase fragment with a polyadenylation signal, followed by a floxed neomycin resistance (neo) cassette, into the C-terminus of the endogenous sphingosine-1-phosphate receptor 1 (S1pr1) gene. This construct was electroporated into (C57BL/6NTac x 129S6/SvEvTac)F1-derived BA1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric mice were bred to EIIa-cre mice (Stock No. 003724) to remove the neo cassette. Offspring were crossed to remove the cre-expressing transgene.

The NLuc-β-arrestin2 targeting construct was designed to insert a floxed neomycin resistance (neo) cassette followed by the N-terminal luciferase fragment, and a polyadenylation signal, into the N-terminus of the β-arrestin2 (Arrb2) gene. This vector was inserted in the first intron of the Gt(ROSA)26Sor locus via electroporation into (C57BL/6NTac x 129S6/SvEvTac)F1-derived BA1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric mice were bred to EIIa-cre mice (Stock No. 003724) to remove the neo cassette.

S1P₁-CLuc mice were bred to NLuc-β-arrestin2 mice to create these S1P₁ luciferase signaling mice. These mice were backcrossed to albino C57BL/6J mice (Stock No. 000058) for at least 4 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize albino C57BL/6J oocytes.

Allele

Symbol: Gt(ROSA)26Sor^{tm2.1(luc*/Arrb2*)Rlp}
Name: targeted mutation 2.1, Richard L Proia
MGI accession ID: MGI:6315195
Generation method: Targeted
Attributes: Reporter
Synonyms: NLuc-beta-arrestin2
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (C57BL/6NTac x 129S6/SvEvTac)F1
Molecular note: The targeting construct is designed to insert a floxed neomycin resistance (neo) cassette followed by the N-terminal luciferase fragment (amino acids 1-416) into the N-terminus of the Arrb2 gene. Cre-mediated recombination removed the upstream floxed neo cassette.
General note: ES cell line BA1

Allele

Symbol: Tyr^c-2J
Name: albino 2 Jackson
MGI accession ID: MGI:1855985
Generation method: Spontaneous
Attributes: Null/Knockout
Marker symbol: Tyr
Marker name: tyrosinase
Marker synonyms: ATN; CMM8; Oca1; OCA1A; OCAIA; SHEP3; skc35
Chromosome: 7
Strain of origin: B6.Cg-Tyrp1^b Hps1^ep
Site of expression: Expressed in melanosomes
Molecular note: This mutation has a G-to-T base change at coding nucleotide 230 resulting in an amino acid change from arginine to leucine at residue 77 (p.R77L) which lies in the highly conserved DDRE sequence. Nucleotide 230 is at the alternative 5' splice donor site for exon 1 and this allele does not produce the 1a or 1b subset of tyrosinase transcripts but does produce a significant increase in 1c and 1d transcripts. Western blots of homozygous mutant skin extracts demostrate the nearly complete absence of the broad 76-84 kDa band of glycosylated wild-type tyrosinase. No tyrosinase activity was found in hairbulb extracts from homozygous mice.

Allele

Symbol: S1pr1^tm4.1Rlp
Name: targeted mutation 4.1, Richard L Proia
MGI accession ID: MGI:6315193
Generation method: Targeted
Attributes: Reporter
Synonyms: S1P1-CLuc
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: (C57BL/6NTac x 129S6/SvEvTac)F1
Site of expression: Highly enriched in endothelial cells.
Molecular note: The targeting construct is designed to insert the C terminal portion of S1pr1 fused to a C-terminal luciferase fragment (amino acids 394-550) with a polyadenylation signal, followed by a floxed neomycin resistance (neo) cassette, into the C-terminus of the S1pr1 gene. Cre-mediated recombination removed the floxed neo cassette.
General note: ES cell line BA1