B6N.129S6(FVB)-S1pr1^{tm3.1(tTA,-Arrb2)Rlp}/J

Stock number: 026275
Research areas: Research Tools

Description

S1pr1 knockin mice use a tobacco etch virus mediated cleavage for identifying sites of S1P1 activation.

Detailed description

S1pr1 knockin mice have a bicistronic transcription unit inserted at the C-terminus of exon 2 of the sphingosine-1-phosphate receptor 1 (S1pr1) gene. Specifically, the allele contains two fusion proteins, a tetracycline-regulated transactivator (tTA)-tobacco etch virus (TEV) protease recognition sequence (tevs) fusion protein and an Arrb2 (murine β-arrestin)-TEV protease fusion protein, separated by an internal ribosome entry site (IRES). Homozygous mice are viable and fertile. S1PR1 is a G protein-coupled receptor (GPCR) that acts as a regulator of vascular development and function in endothelial cells, and has been shown to play a role in autoimmunity and inflammation. In this strain, S1PR tethers tTA to the membrane. Ligand activation of S1PR1 results in phosphorylation of the receptor and subsequent recognition of the tevs sequence by the β-arrestin-TEV protease fusion protein, triggering the release of tTA. Free tTA is then able to enter the nucleus where it regulates transcription of genes of interest under the regulatory control of a tetracycline-responsive promoter element (TRE; tetO). The transcriptional regulation elicited by tTA is blocked by the administration of the tetracycline analog, doxycycline.

When mated to STOCK Tg(tetO-HIST1H2BJ/GFP)47Efu/J transgenic mice (Stock No. 005104), resulting S1P1 GFP mice exhibit tTA-induced dox-dependent green fluorescent protein expression in cells where S1P1 signaling pathway is activated. Fluorescence is evident in thymi, spleen, lymph nodes, lungs, and heart.

Development

A targeting vector was designed with two fusion proteins separated by an internal ribosome entry site (IRES). One fusion protein contains a tobacco etch virus (TEV) protease recognition sequence (tevs) fused to a tetracycline-regulated transactivator (tTA or "Tet-Off") sequence. The second contains a murine arrestin, beta 2 (Arrb2) gene fused to the TEV protease. This construct was inserted at the 3’ end of exon 2 of the sphingosine-1-phosphate receptor 1 (S1pr1) gene and was followed by a polyadenylation sequence and a loxP-flanked neomycin (neo) resistance cassette. This construct was electroporated into 129S6/SvEvTac-derived TC1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric mice were bred with C57BL/6NCr mice. These S1pr1 knockin mice were bred to B6.FVB-Tg(EIIa-cre)C5379Lmgd/J transgenic mice (Stock No. 003724) to remove the neo cassette. Progeny were crossed to remove the cre-expressing transgene, and mice were subsequently bred to C57BL/6NCr mice for at least 7 generations. Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6NJ inbred mice (Stock No. 005304) for one generation.

Allele

Symbol: S1pr1^{tm3.1(tTA,-Arrb2)Rlp}
Name: targeted mutation 1, Richard L Proia
MGI accession ID: MGI:5588124
Generation method: Targeted
Attributes: Inserted expressed sequence; Transactivator
Marker symbol: S1pr1
Marker name: sphingosine-1-phosphate receptor 1
Chromosome: 3
Strain of origin: 129S6/SvEvTac
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli; Arrb2,arrestin, beta 2,mouse, laboratory
Site of expression: Endothelial cells.
Molecular note: A targeting vector was designed with two fusion proteins separated by an internal ribosome entry site (IRES). One fusion protein contains a tobacco etch virus (TEV) protease recognition sequence (tevs) fused to a tetracycline-regulated transactivator (tTA or "Tet-Off") sequence. The second contains a murine arrestin, beta 2 (Arrb2) gene fused to the TEV protease. This construct was inserted at the 3 end of exon 2 of the sphingosine-1-phosphate receptor 1 (S1pr1) gene and was followed by a polyadenylation sequence and a loxP-flanked neomycin (neo) resistance cassette. Cre-mediated recombination removed the selection cassette.