The PDGFRα(S)J conditional knockin allele has the endogenous PDGFRα sequences replaced with a floxed-STOP cassette upstream of the constitutively active PDGFRαJ mutant isoform (V561D mutation). Following Cre-mediated excision of the floxed-STOP cassette, expression of the constitutively active mutant PDGFRα leads to high basal kinase activity without the addition of ligand. These mutant mice may be useful in studying PDGFR signaling in connective tissue development and homeostasis, systemic fibrosis diseases and human systemic sclerosis. As the PDGFRαJ is the most common PDGFRα juxtamembrane domain mutation found in human gastrointestinal stromal tumors (GISTs), these mice may also be used to investigate tumor-suppression in attenuating fibrotic diseases.
Dr. Philippe Soriano, Mount Sinai School of Medicine
The PDGFRα(S)J conditional knockin allele has the endogenous PDGFRα sequences replaced with a loxP-flanked PGKneo (lox-stop-lox) cassette upstream of the constitutively active PDGFRαJ mutant isoform. This V561D mutation in the juxtamembrane domain disrupts inhibitory contacts between the juxtamembrane and kinase domains, which are important for full auto-inhibition, and results in constitutive PDGFRα activity. Prior to Cre recombinase exposure, no expression of the constitutively active αJ mutant isoform is observed, and the mutant allele functions as a knockout. Mice homozygous for the PDGFRα(S)J allele die during embryonic development. Heterozygous PDGFRα(S)J mice are viable and fertile with no reported abnormalities. Following Cre recombinase-mediated excision of the floxed-STOP cassette, high basal kinase activity without the addition of ligand is observed in the cre-expressing tissues.
Of note, pan-deletion of the floxed-STOP cassette during development can be achieved by breeding PDGFRα(S)J conditional knockin mice with Meox2Cre mice expressing Cre recombinase in the early embryo (Stock No. 003755). Alternatively, widespread deletion of the floxed-STOP cassette in adult mice can be achieved by breeding PDGFRα(S)J conditional knockin mice with ROSA26CreER mice ubiquitously expressing a tamoxifen-inducible Cre recombinase (Stock No. 004847). Following tamoxifen treatment in the double mutant embryos/animals, widespread expression of the constitutively active PDGFRαJ leads to connective tissue hyperplasia in embryos and widespread organ fibrosis in adult mice (resembling human autoimmune disease systemic sclerosis). Organs affected are intestine (w polyps), skin, heart, skeletal muscle, kidney glomeruli and renal insterstitium. Vascular smooth muscle is not affected. When comparing widespread Cre recombinase-activated phenotypes of PDGFRαJ to PDGFRαK (see Stock No. 018433), PDGFRαJ expression results in a more attenuated fibrotic disease than PDGFRαK.
To create the PDGFRα(S)J targeting vector, Dr. Philippe Soriano (Mount Sinai School of Medicine) isolated a full-length mouse platelet derived growth factor α (Pdgfra) cDNA sequence and introduced a valine to aspartic acid substitution at codon 561 (V561D) of the juxtamembrane domain by site-directed mutagenesis. This αJ mutation results in a constitutively active mutant PDGFRα and is the most common PDGFRα juxtamembrane mutation found in human gastrointestinal stromal tumors (GISTs). A loxP-flanked PGKneo cassette (lox-stop-lox) was placed between the splice acceptor and the initiating codon of the mutant cDNA sequence. This construct replaced the endogenous Pdgfra exons 2-4 via electroporation into 129S4/SvJaeSor-derived AK7 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and the resulting chimeric males were mated with females to generate the PDGFRα(S)J knockin mouse line. Heterozygous mice were bred to B6;129 mice harboring other mutations/transgenes, and/or to C57BL/6J wildtype, and/or C57BL/6NTac wildtype mice for several generations prior to sending to The Jackson Laboratory Repository. The donating investigator reports selecting PDGFRα(S)J mice with no other mutations present for breeding. Upon arrival, mutant mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish The Jackson Laboratory Repository colony.
|Allele Name||targeted mutation 13, Philippe Soriano|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Constitutively active, Humanized sequence)|
|Gene Symbol and Name||Pdgfra, platelet derived growth factor receptor, alpha polypeptide|
|Strain of Origin||129S4/SvJaeSor|
|Molecular Note||Exons 2 through 4 were replaced with a cDNA of the gene with nucleotide substitutions that result in an amino acid substitution of aspartic acid for valine at position 561 (V561D) preceded by a floxed neo-STOP cassette. This mutation is found in human patients with gastrointestinal stromal tumors and leads to a constitutively active form by disrupting the inhibitory conotacts between the juxtamembrane and kinase domain. Removal of the floxed neo-STOP cassette is necessary to achieve expression.|
|Mutations Made By|| |
Dr. Philippe Soriano, Mount Sinai School of Medicine
When maintaining a live colony, heterozygous mice may be bred to wildtype mice from the colony. Homozygous mice die during embryonic development.
When using the STOCK Pdgfratm13Sor/J mouse strain in a publication, please cite the originating article(s) and include JAX stock #018432 in your Materials and Methods section.