B6(Cg)-Pah^{tm1.1(PAH*R408W)Xiwan}/J

Stock number: 039069
Product name: exon humanized PAH R408W
Research areas: Metabolism Research, Mouse/Human Gene Homologs, Neurobiology Research

Description

Pah exon humanized R408W knock-in mice carry the human PAH exon 12 with a R408W (arginine to tryptophan) mutation inserted into the Pah (phenylalanine hydroxylase) gene. The homologous human mutation R408W is associated with phenylketonuria (PKU). Homozygous mice are hypopigmented and have elevated blood phenylalanine levels. This strain maybe useful for studies of PKU and for humanized therapeutics testing.

Of note, the humanized Pah P281L variant is available as Stock No. 039067.

Detailed description

Pah (phenylalanine hydroxylase) hydroxylates phenylalanine to tyrosine and is the rate-limiting step in phenylalanine catabolism, mutations in Pah are associated with phenylketonuria (PKU). High levels of blood phenylalanine (more than 1200 μmol/L) lead to progressive neurological symptoms and seizures. In humans, the most frequently occurring pathogenic variant worldwide is R408W (arginine to tryptophan at position 408).

Pah knock-in mice carry PAH human exon 12 with an amino acid substitution at position 408 altering arginine to tryptophan (R408W). Mice homozygous for this knock-in mutation are viable and fertile, but homozygous females have smaller and fewer litters and pups generally die perinatally. Homozygotes have elevated blood phenylalanine levels, exhibit hypopigmentation due to reduced melanin synthesis, and are smaller in size. Blood phenylalanine levels range from approximately 1000-1500 μmol/L as compared to 120 μmol/L in controls (similar to humans).

Of note, the humanized Pah P281L variant is available as Stock No. 039067.

Development

Homologous recombination in C57BL/6 mouse embryonic stem cells was used to replace exon 12 with the orthologous human PAH exon 12 sequence including 500 bp of flanking genomic sequence both 5' and 3' of exon 12, exon 12 also contains the amino acid substitution at position 408 altering arginine to tryptophan (R408W; c.1222 C to T) of the phenylalanine hydroxylase (Pah) gene on chromosome 10. The construct also included a loxP-flanked neomycin selection cassette upstream of exon 12. Pah transcript Pah-201 (ENSMUSG0000020241.17) was used as reference for the exon number. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric males were bred with C57BL/6J mice. Mice were then crossed to mice carrying a ubiquitous unspecified Cre recombinase on the C57BL/6 background (Ozgene) to remove the neomycin cassette. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

In 2024, 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 males sent to The Jackson Laboratory Repository. Two of the 43 markers throughout the genome, on Chromosomes 9 and 13, were segregating with an unknown source. Also, 2 of the 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/6N;C57BL/6J genetic background.

Allele

Symbol: Pah^{tm1.1(PAH*R408W)Xiwan}
Name: targeted mutation 1.1, Xiao Wang
MGI accession ID: MGI:7574743
Generation method: Targeted
Attributes: Humanized sequence
Synonyms: PAH c.1222CT
Marker symbol: Pah
Marker name: phenylalanine hydroxylase
Marker synonyms: PH; PKU; PKU1
Chromosome: 10
Strain of origin: C57BL/6J
Molecular note: Homologous recombination in C57BL/6 mouse embryonic stem cells was used to replace exon 12 with the orthologous human PAH exon 12 sequence including 500 bp of the flanking genomic sequence of both 5' and 3' of exon 12. Additionally, exon 12 also contains the amino acid substitution at position 408 altering arginine to tryptophan (R408W; c.1222 C to T) of the phenylalanine hydroxylase (Pah) gene on chromosome 10. The construct also included a loxP-flanked neomycin selection cassette upstream of exon 12 which was subsequently removed by crossing to Cre deleter.