• The PAH enzyme functions in liver as homo-tetramers to hydroxylate phenylalanine (Phe) into tyrosine and requires the co-factor tetrahydrobiopterin for catalysis. Phenylalanine hydroxylase (PAH) deficiency prevents the conversion of Phe to tyrosine. PAH deficiency results in hyperphenylalaninemia (HPA) and irreversible neurological consequences if left untreated.
• The exons 1-13 of mouse Pah gene that encode the full-length protein were knocked out in B-Pah KO mice.
• Mouse Pah mRNA was exclusively detectable in wild-type mice but not in homozygous B-Pah KO mice.
• PAH protein expression was detected in the liver and kidney of wild mice but not in homozygous B-Pah KO mice.
• B-Pah KO mice provide a powerful preclinical model for further development of gene and cell biologics to treat phenylketonuria (PKU). The serum Phe concentration in homozygous B-Pah KO mice was higher than those in wild-type mice, additionally, Phe levels were further elevated in B-Pah KO mice fed with a standard chow (~2300 μM) compared to those maintained on the low-Phe diet.
• Application: For instance, this product is designed to be incorporated into the existing experimental toolkit for PKU scientists, thereby enhancing the research capabilities and propelling the advancement of PKU research

Gene targeting strategy for B-Pah KO mice. The exons 1-13 of mouse Pah gene that encode the full-length protein were knocked out in B-Pah KO mice.

Strain specific analysis of PAH mRNA expression in wild-type C57BL/6 mice and B-Pah KO mice by RT-PCR. Liver RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-Pah KO mice (-/-), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Pah primers. Mouse Pah mRNA was exclusively detectable in wild-type mice but not in homozygous B-Pah KO mice.

Western blot analysis of PAH protein expression in homozygous B-Pah KO mice. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-Pah KO mice (-/-), and then analyzed by western blot with anti-PAH antibody (abcam, ab178430). 40 μg total proteins were loaded for western blotting analysis. PAH protein expression was only detected in the liver and kidney of wild-type mice but not in the homozygous B-Pah KO mice.

Hair coat color and body weight in homozygous B-Pah KO mice. (A) When compared to wild-type C57BL/6 mice (+/+, male, 7-week-old), homozygous B-Pah KO mice (-/-, male, 7-week-old) exhibit gray fur. (B) Body weight of homozygous B-Pah KO mice was significantly lower than that of wild-type control mice. Values are expressed as mean ± SEM. Significance was determined by unpaired t test.  *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Phenylalanine concentration analysis in homozygous B-Pah KO mice. (A). At 10 weeks of age, three male and three female homozygous B-Pah KO mice were switched from a low-phenylalanine (low-Phe) diet to standard chow for one week, while an additional three male and three female KO mice remained on the low-Phe diet throughout the study. Wild-type control mice were maintained on standard chow continuously. (B). Serum phenylalanine (Phe) concentration was measured using phenylalanine assay kit (abcam, ab241000). The serum Phe concentration in homozygous B-Pah KO mice was higher than those in wild-type mice, additionally, Phe levels were further elevated in B-Pah KO mice fed with a standard chow (~2300 μM) compared to those maintained on the low-Phe diet. Values are expressed as mean ± SEM. Significance was determined by unpaired t test.  *P < 0.05, **P < 0.01, ***P < 0.001 , ****P < 0.0001.