B6.129S6(D2)-Ptger4^tm1.1Matb/BreyJ

Stock number: 028102
Product name: EP4flox
Research areas: Cardiovascular Research, Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Reproductive Biology Research, Research Tools

Description

These floxed mutant mice possess loxP sites flanking exon 2 of the Ptger4 gene. This strain may be useful for generating conditional mutations in studies of the role of prostaglandin E2 (PGE2) and function of the PGE2 EP4 receptor.

Detailed description

Prostaglandin E2 (PGE2), a fatty acid derivative and a primary target of NSAIDs, is important in diverse physiological processes, including but not limited to: contraction and relaxation of smooth muscle, vasodilation, vasoconstriction, blood pressure regulation, immune response and inflammation regulation and bone formation and healing. There are four receptors for PGE2. The Ptger4 gene encodes a G-protein coupled PGE2 receptor (EP4), which is essential for embryonic development and neonatal survival in mice. These mice possess loxP sites on either side of exon 2 of the targeted gene. Mice that are homozygous for this allele are viable and fertile. When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have exon 2 deleted in the cre-expressing tissues. Removal of the floxed sequence creates a null allele. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background.

When bred to a strain with Cre recombinase expression in the myeloid lineage, this mutant mouse strain may be useful in studies of innate immunity and inflammation in the CNS.

Development

A targeting vector designed by Dr. Matthew Breyer (Vanderbilt University) containing a loxP site flanked PGK-Neo selection cassette was utilized in the construction of this mutant. This selection cassette was inserted downstream of exon 2 of the targeted gene, and another loxP site was inserted upstream of exon 2. This construct was electroporated into 129S6/SvEvTac derived TL-1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting male EP₄^lox+neo chimeric animals were bred to C57BL/6 females. Heterozygous EP₄^lox+neo mice were intercrossed to generate homozygotes. Mice homozygous for the EP₄^lox+neo allele die perinatally due to patent ductus arteriosus. Heterozygous EP₄^lox+neo male mice were then bred to B6D2 F1 females to produce fertilized oocytes, which were subsequently transfected with a Cre recombinase plasmid. The resulting single cell embryos were transferred to pseudopregnant females. The male offspring that was mosaic for all 3 alleles was bred to C57BL/6 females to produce heterozygous EP₄^flox mice. Heterozygous EP₄^flox were intercrossed to obtain homozygotes. The donating investigator reported that mice were then backcrossed to C57BL/6J for 10 generations (see SNP note below). During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background. 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).

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. One of the 27 markers throughout the genome was segregating with 129 or DBA on Chromosome 15. Also, 4 of 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 mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Ptger4^tm1.1Matb
Name: targeted mutation 1.1, Matthew D Breyer
MGI accession ID: MGI:3052967
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Ptger4
Marker name: prostaglandin E receptor 4 (subtype EP4)
Chromosome: 15
Strain of origin: 129S6/SvEvTac
Molecular note: The neo cassette was removed via partial cre recombination leaving loxP sites surrounding exon 2, which encodes slightly more than five of the seven transmembrane-spanning domains. Recombination in mutant mice was confirmed by Southern blot and PCR.