B6.129S4-Pdgfra^tm12Sor/J

Stock number: 018433
Product name: PDGFRα^(S)K
Strain synonyms: lox-stop-lox αK , a(S)K
Research areas: Cancer Research, Cardiovascular Research, Cell Biology Research, Dermatology Research, Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools

Description

The PDGFRα^(S)K conditional knockin allele has the endogenous PDGFRα sequences replaced with a floxed-STOP cassette upstream of the constitutively active PDGFRαK mutant isoform (D842V mutation in the kinase domain). Following Cre recombinase-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 systemic fibrosis diseases and human systemic sclerosis. As the PDGFRαK is the single most common PDGFRα mutation found in human gastrointestinal stromal tumors (GISTs) and PDGFRα kinase domain mutations are associated with lymphomas, leukemias, and testicular tumors, these mice may also be used to investigate such disease and tumor-suppression in attenuating fibrotic diseases.

Detailed description

The PDGFRα^(S)K conditional knockin allele has the endogenous PDGFRα sequences replaced with a loxP-flanked PGKneo (lox-stop-lox) cassette upstream of the constitutively active PDGFRαK mutant isoform. This D842V mutation in the kinase domain interferes with the inactive conformation of the ATP-binding pocket leading to constitutive PDGFRα activity. Prior to Cre recombinase exposure, no expression of the constitutively active αK mutant isoform is observed, and the mutant allele functions as a knockout. Mice homozygous for the PDGFRα^(S)K allele die during embryonic development. Heterozygous PDGFRα^(S)K 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)K conditional knockin mice with Meox2^Cre 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)K conditional knockin mice with ROSA26^CreER 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 PDGFRαK 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 PDGFRαK to PDGFRαJ (see Stock No. 018432), PDGFRαK expression results in greater fibrotic disease than PDGFRαJ.

Development

To create the PDGFRα^(S)K 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 an aspartic acid to valine substitution at codon 842 (D842V) of the kinase domain by site-directed mutagenesis. This αK mutation results in a constitutively active mutant PDGFRα and is the single most common PDGFRα 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)K knockin mouse line. Heterozygous mice were bred to C57BL/6J wildtype and/or C57BL/6NTac wildtype mice for at least eight generations prior to sending to The Jackson Laboratory Repository. 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

Symbol: Pdgfra^tm12Sor
Name: targeted mutation 12, Philippe Soriano
MGI accession ID: MGI:3838504
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Constitutively active; Humanized sequence
Marker symbol: Pdgfra
Marker name: platelet derived growth factor receptor, alpha polypeptide
Chromosome: 5
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 valine for aspartic acid at position 842 (D842V) 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 interfering with the inactive conformation of the ATP-binding pocket. Removal of the floxed neo-STOP cassette is necessary to achieve expression.