129S4-Pdgfra^tm5Sor/J

Stock number: 028663
Product name: PDGFα^P13K
Research areas: Developmental Biology Research

Description

This PDGFRα^PI3K knockin strain carries a point mutation in the PI3K binding site. These mice may be suitable for use in studies related to development and the PI3K and AKT1 signaling pathways.

Detailed description

The cell surface tyrosine kinase receptor PDGFRA (platelet derived growth factor receptor, alpha polypeptide) is critical in embryonic development (in particular, skeletal development and neural tube closure), and in cell proliferation, migration, and differentiation. Mutations in and fusion genes including the human PDGFRA gene are associated with cancers such as chronic myeloid leukemia, hematolymphoid neoplasm, and gastrointestinal stromal tumors. These mice carry a point mutation in the PI3K (phosphatidylinositol 3-kinase) binding site. PI3K is the major downstream effector protein of PDGFRA.

Mice that are heterozygous for the knock-in targeted mutation are viable and fertile. Homozygotes exhibit a neonatal lethal phenotype: most homozygotes die just after birth, any surviving homozygotes die by postnatal day 16. Expression of PDGFRA receptor protein in homozygotes is similar to wildtype control levels as detected by Western blots of knock-in mutant embryo and primary mesenchymal cell lysates. Homozygotes that survive past the neonatal stage are 40% smaller in size than wildtype controls and exhibit cleft palate with incomplete penetrance (71%). Newborn homozygotes have an emphysema-like lung phenotype with failed alveolar septation, and abnormal skeletal development (abnormal cervical vertebrae, abnormal shoulder girdle, spina bifida, failed sternum closure). Hypomyelination is observed in the cerebellum, corpus callosum and spinal cord, but not in all regions of the CNS. Homozygous E18.5 embryos display defective somite-derived cell migration and impaired lumbar vertebral arch formation. Akt (protein kinase B)is not phosphorylated in homozygous animals. Heterozygous embryos have fewer oligodendrocyte progenitor cells compared with wildtype controls and exhibit impaired oligodendrocyte progenitor cell migration. These mice still carry the neo selection cassette.

Development

Site-directed mutagenesis was utilized to introduce tyrosine to phenylalanine mutations at Tyr-731/742 in Pdgfra cDNA. A targeting vector containing this Pdgfra cDNA encoding a mutated receptor with point mutations at tyrosine 731/742 residues (in the PI3K binding site), and a floxed PGKneo selection cassette was electroporated into 129S4/SvJaeSor derived AK7 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. Although the mice were bred to a germline Cre deleter strain, MORE (Meox2-Cre), on the 129S4/SvJaeSor genetic background, to excise the PGKneo cassette, it was found that the mice still carry the PGKneo cassette.

The mice were then backcrossed to 129S4/SvJaeSor for more than 20 generations.

Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize 129S4/SvJaeJ oocytes (Stock No. 009104). Of note, these mice still carry the neo selection cassette.

Allele

Symbol: Pdgfra^tm5Sor
Name: targeted mutation 5, Philippe Soriano
MGI accession ID: MGI:2656146
Generation method: Targeted
Attributes: Hypomorph; Inserted expressed sequence
Marker symbol: Pdgfra
Marker name: platelet derived growth factor receptor, alpha polypeptide
Chromosome: 5
Strain of origin: 129S4/SvJaeSor
Molecular note: A cDNA encoding a mutated receptor consisting of point mutations encoding the tyrosine 731/742 sites was targeted into the Pdgfra locus with a loxP-flanked neomycin cassette. Western blot analysis indicated that expression of the targeted Pdgfra locus was disrupted and the mutant receptor was expressed.