btbr pah enu2 mice  (Jackson Laboratory)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Rational design and applications of piperazine and cyclohexane ionizable lipids for PKU and SSADH deficiency

doi: 10.1016/j.omtn.2025.102697

Figure Lengend Snippet: Enzyme replacement therapy for PAH deficiency via mRNA/LNP delivery (A) Schematic representation of the enzyme replacement therapy after PAH mRNA/LNP delivery. (B and C) In vitro PAH protein expression (B) and PAH activity (C) were evaluated in hPAH mRNA-MC3 LNP-treated HEK293T cells. (D) Phe levels were measured in serum from Pah enu2 mice following a single i.v. dose of hPA H- mRNA-MC3 ( n = 2–3/group, mean ± SD). (E–G) All experiments were performed using unmodified hPAH - mRNA synthesized with wild-type UTP. (E) In vitro PAH expression in hPAH - mRNA-GCP LNP-treated HEK293T cells. The data are presented as the expression relative to the expression of hPAH -mRNA in MC3-treated HEK293T cells ( n = 2/group, mean ± SD). (F) In vivo PAH protein expression was evaluated by western blotting in normal (WT) mice at 6 and 24 h after a single i.v. injection of either vehicle or 1 mg/kg hPAH - mRNA/LNP ( n = 3/group, mean ± SD). (G) Serum Phe levels were determined at multiple time points (before and 6, 24, and 48 h after dosing) in Pah enu2 mice ( n = 3–5/group, mean ± SD) following a single i.v. dose of hPAH -mRNA-LNPs. The dotted line indicates the target concentration of serum Phe. Data are represented as the mean ± standard deviation. The data were analyzed by the Kruskal-Wallis test with Dunn’s multiple comparisons test.

Article Snippet: BTBR-Pah enu2 mice are a model of heritable PKU generated by ethylnitrosourea (ENU)-induced mutagenesis, leading to a missense mutation in exon 7, and mice were obtained from the Jackson laboratory (Strain#002232).

Techniques: In Vitro, Expressing, Activity Assay, Synthesized, In Vivo, Western Blot, Injection, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Structure-guided development of a potent human B 0 AT1 inhibitor effective in a mouse model of phenylketonuria

doi: 10.1101/2025.10.27.684712

Figure Lengend Snippet: a) Schematic of the in vivo study design. Pah enu2 mice were orally administered compound 3 (30 mg/kg), and blood and urine samples were collected after 4 hours post-dose. b) Urinary excretion of phenylalanine (Phe) measured 4 hours after compound 3 administration. Mice were placed in metabolic cages immediately after dosing, and urine was collected over 4 hours. c) Plasma Phe concentrations measured 4 hours after oral administration of compound 3. Phe levels were quantitated by LC–MS/MS. Data are shown as mean ± s.e.m. (n = 8 mice per group). Each dot represents an individual mouse. **p < 0.01 compared to vehicle group (Student’s t-test)

Article Snippet: Male Pah enu2 mice (12 weeks old, The Jackson Laboratory Japan, Inc., Yokohama, Japan) were used as test animals.

Techniques: In Vivo, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A and B ) Increased Phe concentration in the serum ( A ) and CSF ( B ) of adult Pah Enu2 mouse. ( C ) Experimental design for NMDAR-EPSC (top) and I NMDA (bottom) recordings. Stim., stimulating electrode; Rec., recording electrode. ( D ) Representative traces of NMDAR-EPSCs (top) obtained at the indicated time points ( , ), and the time course of the peak amplitudes (bottom left) of NMDAR-EPSCs measured at −40 mV in CA1 neurons. Bottom right: l -Phe had no effect on the peak amplitudes of NMDAR-EPSCs. ( E ) In the presence of NBQX and picrotoxin, I NMDA was induced by bath application of NMDA (5−10 μM). Different concentrations of l -Phe were perfused with NMDA for 5 minutes. ( F ) l -Phe exhibits concentration-dependent bidirectional effects on I NMDA . ( G ) I NMDA was induced by 3−12 μM NMDA in the presence of GluN2A or GluN2B blockers. ( H ) l -Phe-induced facilitation of I NMDA was blocked by Ro (2 μM) or ifen (6 μM) but not by PEAQX (0.5 μM). ( I and J ) A sample trace (left) and summary (right) of I NMDA measured before and during l -Phe perfusion in HEK293 cells expressing GluN2A ( I ) or GluN2B ( J ). ( K ) Representative traces (top) and the peak amplitudes (bottom left) of NMDAR-EPSCs measured in the presence of TBOA (10 μM). l -Phe induced facilitation of NMDAR-EPSCs in each condition (bottom right). ( L ) Addition of 5, 10, and 20 μM glycine attenuated l -Phe-induced I NMDA facilitation. I NMDA was induced by 5 μM NMDA. ( M ) The concentration relationship between l -Phe-induced facilitation of I NMDA and added glycine (Gly) concentration. A Student’s t test ( A , B , D , F , I , and J ) or a 1-way ANOVA with a post hoc Tukey’s test ( H and K ) was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, P ≥ 0.05. Scale bars: 50 ms and 50 pA ( D and K ) Veh, vehicle.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Concentration Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A – D ) Representative Western blots and expression levels of NMDAR subunits in the total ( A and B ) and synaptosomal fractions ( C and D ) of WT and Pah Enu2 hippocampi. ( E ) Representative AMPAR- and NMDAR-EPSCs measured at −70 and +40 mV, respectively, in CA1 pyramidal neurons. ( F ) The peak amplitudes of NMDAR-EPSCs were plotted against AMPAR-EPSCs. ( G ) Normal AMPA to NMDA ratios in the CA1 pyramidal cells of Pah Enu2 mice. ( H ) Bath application of NMDA (5 μM) induced similar magnitudes of inward current in CA1 pyramidal neurons of WT and Pah Enu2 mice. I NMDA was measured in the presence of NBQX and picrotoxin. PEAQX (0.5 μM) reduced I NMDA , and subsequent AP-5 (50 μM) perfusion blocked the remnant. ( I ) Total I NMDA and PEAQX-sensitive and -insensitive components after sequential application of PEAQX and AP-5 in CA1 pyramidal cells. ( J ) Hippocampal expression levels (bottom) of α5-GABA A R were determined by Western blotting (top). ( K ) Representative traces of tonic currents measured from WT and Pah Enu2 hippocampal CA1 neurons in the presence and absence of Phe. Vehicle (Veh) or Phe (250 μM) were perfused throughout the recordings. Scale bar: 30 seconds and 100 pA. ( L ) Magnitudes of bicuculline (40 μM)-sensitive tonic current in each condition are summarized. Statistical analysis was performed using Student’s t test ( B , D , G , I , and J ) and 2-way ANOVA ( L ). ** P < 0.01 and NS, P ≥ 0.05.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A ) l -Phe reduces the magnitude of LTP in both WT and Pah Enu2 mice. Top: representative traces of fEPSP obtained at the indicated time points. Bottom left: fEPSP slopes were normalized to those obtained in the baseline and plotted against time. l -Phe was perfused from 5 minutes before to 1 minutes after TBS (arrow). Bottom right: fEPSP slopes during the last 10 minutes were normalized to baseline. ( B ) GluN2B antagonists block the effect of l -Phe on the TBS (arrow)-induced LTP. Sample traces (top), time course of fEPSP slopes (bottom left), and the magnitude of LTP (bottom right) in each condition. ( C and D ) PEAQX blocks LTP induction, and l -Phe perfusion during the peri-TBS period induces an LTD-like decrease in fEPSP slopes. ( E and F ) l -Phe and TBS had no effect on the slope of fEPSPs under GluN2A and GluN2B inhibition. ( G − K ) Representative Western blots ( G ), and the ratios of phosphorylated GluA1-S845 to total GluA1 ( H ), total GluA1 to -tubulin ( I ), phosphorylated GluA2-S880 to total GluA2 ( J ), and total GluA2 to -tubulin ( K ) in the CA1 region of acute hippocampal sections harvested 30 minutes after TBS. ( L ) WT and Pah Enu2 sections exhibit similar synaptic responses to LFS. ( M ) Normalized fEPSP slopes during the last 10 minutes in WT and Pah Enu2 sections. ( N ) Ro blocks the effect of l -Phe on LTD facilitation. Sample traces of fEPSPs (top). ( O ) Normalized fEPSP slopes during the last 10 minutes in each condition. ( A − C , E , L , and N ) Scale bars: 5 ms and 0.5 mV. Statistical analysis was performed using Student’s t test ( D , F , and M ), 1-way ( H − K , and O ) or 2-way ANOVA ( A ), or Kruskal-Wallis test ( B ) with post hoc Tukey’s test. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, P ≥ 0.05. Con, control; Veh, vehicle.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Blocking Assay, Inhibition, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A and B ) Phe concentrations in the serum ( A ) and CSF ( B ) were measured 30 minutes after vehicle (Veh) or l -Phe (1 mg/g, i.p.) administration. ( C ) Western blot analyses for the protein levels of p-eEF2 and eEF2 in Veh- and l -Phe–treated mice. Whole brains (WB) and hippocampi (HP) were collected 30 minutes after Veh or l -Phe (1 mg/g, i.p.) administration. Bottom: Quantification of the p-eEF2 to eEF2 ratio. ( D ) Enhanced eEF2 phosphorylation (top) and increased p-eEF2 to eEF2 ratio (bottom) in the Pah Enu2 hippocampus. ( E ) Experimental design for fiber photometry recording in the mPFC of Camk2a-Cre mice. Bottom: immunohistochemical staining of an mPFC section showing the GCaMP6s-expressing cells (green) and canula placement. DAPI (blue) was used to identify the brain structures. Calibration, 200 μm. ( F ) Neuronal activity of CaMKII-expressing cells in the mPFC was decreased by l -Phe administration. The bottom panel shows the ΔF/F signals obtained during the indicated periods (1, 2, and 3) on an expanded time scale. ( G ) Quantification of the frequency of Ca 2+ transients obtained from 5 mice. ( H ) Ro blocks l -Phe–induced eEF2 phosphorylation in the hippocampus. Ro (3 mg/kg, i.p.) was administered 30 minutes before l -Phe or vehicle injection. ( I ) l -Phe and Ro were sequentially administered to Camk2a-Cre mice during the recording. ( J ) Summary of the frequency of F transients during the baseline and perfusion of l -Phe and Ro. Statistical analysis was performed using the Mann-Whitney U test ( A ), Student’s t test ( B − D and H ), or 1-way ANOVA with post hoc Tukey’s test ( G and J ). * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, P ≥ 0.05.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Western Blot, Phospho-proteomics, Immunohistochemical staining, Staining, Expressing, Activity Assay, Injection, MANN-WHITNEY

Journal: Experimental Biology and Medicine

Article Title: Gender difference in pre-clinical liver-directed gene therapy with lentiviral vectors

doi: 10.3389/ebm.2025.10422

Figure Lengend Snippet: Differential transduction efficiencies in male vs. female Pah enu2 mice dosed IV with either OXB-401 or OXB-Null. Median VCN is shown for each group at day 85 post dosing. No significant differences were observed between the groups (p > 0.05).

Article Snippet: For the second study, BTBR- Pah enu2 /J mice [ ], also referred to as Pah enu2 ) were obtained from The Jackson Laboratory and dosed IV with TSSM buffer (200 μL), OXB-Null (4 × 10 10 TU/kg) or OXB-401 (4 × 10 10 TU/kg) and observed for the duration of the study (85 days).

Techniques: Transduction

Journal: Communications Biology

Article Title: Hyperphenylalaninemia and serotonin deficiency in Dnajc12 -deficient mice

doi: 10.1038/s42003-024-07360-6

Figure Lengend Snippet: a Representative western blot for PAH in the liver of DNAJC12 KO and wild-type (WT) mice. b Quantification of PAH expression in the liver of DNAJC12 KO ( n = 7) mice in comparison to WT ( n = 7). GAPDH was used as loading control. GAPDH was used as loading control. Phe concentrations in the liver ( c ) and serum ( d ) of DNAJC12 KO and PAH enu2 (PAH KO) mice relative to wild-type (WT) mice ( n = 4). e 5-HTP in vitro generation assay in the liver of DNAJC12 KO ( n = 5), WT ( n = 5) and TPH1/TPH2-double KO ( n = 2) mice. *, p < 0.05, ** p < 0.01, *** *p < 0.0001. b : Student’s t -test; c–e: One-way ANOVA wi t h Tukey’s multiple comparisons. Data are presented as mean ± SEM.

Article Snippet: PAH enu2 mice (PAH-KO) were obtained from Jackson Laboratories, USA (#002232).

Techniques: Western Blot, Expressing, Comparison, Control, In Vitro

Journal: Scientific Reports

Article Title: Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

doi: 10.1038/s41598-021-02267-2

Figure Lengend Snippet: Effect of PAH and PAL gene transfer on blood Phe and Tyr levels after gene delivery to PAH enu2 mice. (A) Schematic of in-vivo study design. PAH and PAH encoding AAV vectors were administered by one-time IV delivery on Day 0. (B) Blood Phe levels were measured in naïve PAH enu2 mice, PAH enu2 mice administered with PAH (1e11 vg) or PAL (4e10, 1e11 or 3e11 vg/mouse) and HET mice at day 9 (baseline) and days 5, 14, 34 and 41 post treatment. Significant reduction in blood Phe levels was observed in both PAH and PAL in PAH enu2 mice as compared to naïve PAH enu2 mice. PAH enu2 PAL treated mice show lower Phe than HET mice while PAH treated mice maintain Phe levels comparable to HET mice. (C) Significant increase in blood Tyr to levels comparable to HET mice were achieved in PAH treated mice but not in PAL treated mice. Two-way ANOVA Mixed effect analysis was performed, ***p < 0.001. (D) Blood Phe continued to remain low in PAH and PAL PAH enu2 mice until the end of the study. (E) Blood Tyr remained normal in PAH PAH enu2 mice till the end of the study. Treatment groups consisted of n = 8–10 animals. Statistics by One-way ANOVA Tukey’s multiple comparison, ***p < 0.001.

Article Snippet: A colony of PAH-deficient BTBR-PAH enu2 (PKU) mice was maintained at Taconic .

Techniques: In Vivo, Comparison

Journal: Scientific Reports

Article Title: Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

doi: 10.1038/s41598-021-02267-2

Figure Lengend Snippet: Brain amino acid levels post PAH or PAL administration. (A) Brain Phe is reduced to normal levels in PAH or PAL treated PAH enu2 mice. (B) Brain Tyr levels are restored to normal in PAH PAH enu2 mice but not in PAL PAH enu2 mice. (C) Brain Trp is increased in both PAH and PAL treated PAH enu2 mice. N = 5 per group were used for analysis. Control mice and mice treated with 1e11 vg/mouse of PAH or PAL vector were terminated on day 41 and perfused with PBS. Naïve untreated PAH enu2 mice, PAH or PAL , treated PAH enu2 mice, HET untreated HET mice. One-way ANOVA Tukey’s multiple comparison, **p < 0.01 and ****p < 0.0001.

Article Snippet: A colony of PAH-deficient BTBR-PAH enu2 (PKU) mice was maintained at Taconic .

Techniques: Control, Plasmid Preparation, Comparison

Journal: Scientific Reports

Article Title: Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

doi: 10.1038/s41598-021-02267-2

Figure Lengend Snippet: Restoration of brain neurotransmitter levels after PAH or PAL administration. (A) Schematic of major steps involved in synthesis of neurotransmitters dopamine and norepinephrine. ( B) Dopamine precursor L-DOPA was significantly higher in PAL PAH enu2 and HET brains as compared to the naïve PAH enu2 brains. Significant increases in (C) dopamine and (D) norepinephrine were observed in both the PAH and PAL PAH enu2 mouse brains as compared to the naïve mice. (E) Schematic of serotonin synthesis and downstream metabolite 5-hydroxyindolacetic acid (5-HIAA). Serotonin precursor 5-hydroxytryptophan (F) , serotonin (G) and 5-HIAA (H) were all restored to levels similar to HET mice after PAH and PAL administration. N = 5 per group were used for analysis. Control mice and mice treated with 1e11 vg/mouse of PAH or PAL vector were terminated on day 41 and perfused with PBS. Group abbreviations: PKU naïve, untreated PAH enu2 mice; PKU PAH or PAL, treated PAH enu2 mice; HET, untreated HET mice. One-way ANOVA Tukey’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: A colony of PAH-deficient BTBR-PAH enu2 (PKU) mice was maintained at Taconic .

Techniques: Control, Plasmid Preparation, Comparison

Journal: Scientific Reports

Article Title: Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

doi: 10.1038/s41598-021-02267-2

Figure Lengend Snippet: Improvement in nesting behavior in PAH and PAL PAH enu2 mice. (A) Baseline behavior measured 2 days prior to treatment and (B) improved at day 34 post PAH and PAL treatment. (C) Tracking the nesting score of individual mice shows that most PAH and PAL treated mice perform better and receive a higher score in the post treatment nesting assay. Treatment groups consisted of n = 8–10 animals. Group abbreviations: PKU naïve, untreated PAH enu2 mice; PKU PAH or PAL, treated PAH enu2 mice; HET, untreated HET mice. Statistics by One-way ANOVA Kruskal–Wallis test followed by Dunn’s multiple comparison, *p < 0.05 and ***p < 0.001.

Article Snippet: A colony of PAH-deficient BTBR-PAH enu2 (PKU) mice was maintained at Taconic .

Techniques: Comparison

Journal: Scientific Reports

Article Title: Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

doi: 10.1038/s41598-021-02267-2

Figure Lengend Snippet: Gene expression changes in livers of PAH enu2 mice. (A) Number of differentially expressed genes in naïve, PAH and PAL treated PAH enu2 mice as compared to HET mice (HET). (B) Venn diagram showing number of DEG common and unique to each group. The majority of DEG in PAH enu2 mice are corrected after PAH or PAL treatment. (C ) Top 10 pathways affected in the liver of PAH enu2 mice as compared to HET mice analyzed by Ingenuity pathway analysis. Superpathway of cholesterol biosynthesis was the most affected pathway in the liver of PAH enu2 mice. (D) Heatmap showing the expression of genes involved in multistep cholesterol biosynthesis pathway. Most genes involved in cholesterol biosynthesis are upregulated in untreated PAH enu2 mice (PKU naive). The expression profile of PAH and PAL treated mice shares similarity to naïve HET mice (HET, control). Heatmap was generated using QIAGEN Omicsoft studio version 10.2.4.7. (E) Top 6 upregulated and downregulated genes in PAH enu2 mice as compared to naïve HET mice. (F) Modest increase in expression of PPARα, activator of Cyp4a14 and FAT, a downstream effector of high Cyp4a14 in PAH enu2 mice. Upregulation of Cyp4a14, PPARα and FAT is corrected in both PAH and PAL treated mice. N = 3 per group were used for analysis. One-way ANOVA Tukey’s multiple comparison, *p < 0.05 and **p < 0.01.

Article Snippet: A colony of PAH-deficient BTBR-PAH enu2 (PKU) mice was maintained at Taconic .

Techniques: Gene Expression, Expressing, Control, Generated, Comparison

Journal: Scientific Reports

Article Title: Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

doi: 10.1038/s41598-021-02267-2

Figure Lengend Snippet: Changes in protein levels in livers of PAH enu2 mice. (A) Number of differentially expressed proteins in naïve, PAH and PAL treated PAH enu2 mice as compared to HET mice. (B) Top 10 pathways affected in the livers of PAH enu2 mice as compared to HET mice analyzed by Ingenuity pathway analysis. Pathways linked to lipid metabolism are the most affected pathways in the liver of PAH enu2 mice. (C) Top 6 upregulated and downregulated proteins in PAH enu2 mice as compared to HET mice. (D) Network analysis identified Lipid metabolism as the top network in the dataset with Cyp4a11 (Cyp4a10 in mouse) at the bottom of the hierarchy. Network analysis figure was generated using Ingenuity Pathway analysis (IPA) (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis ).

Article Snippet: A colony of PAH-deficient BTBR-PAH enu2 (PKU) mice was maintained at Taconic .

Techniques: Generated