Table 1.
LC-MS detection parameters for NADP+ and NADPH.
Fig 1.
(A) Representation of the wild-type Me1 gene (top) and two Me1 knockout alleles, Me1 null (middle) and Me1 flox (bottom). (B) The Me1 germline knockout model (Me1 KO). (C) The UBC-Cre Me1 conditional knockout model (Me1 cKO). Arrows represent primer locations; presentation is not to scale. (D-F) Genotype validation of (D) the Me1 null lacZ reporter, (E) Me1 flox and wild-type, and (F) UBC-Cre alleles by PCR of tail DNA. The black forward primer and the black reverse primer produce a ~1.5Kbp band for the null allele. The red forward primer and the black reverse primer produce a ~1Kbp band for the floxed allele and a ~750bp band for the wild type allele. The UBC-CreERT2 allele is transgenic.
Fig 2.
(A) Dihybrid cross breeding scheme for mice containing one Me1 null allele and one wild-type allele. (B) Percentage of pups (n = 142) and their corresponding genotypes compared to expected Mendelian ratios and broken down by sex. Non-Mendelian transmission of the Me1 null allele is noted, with a greater than expected number of Me1 KO and heterozygous Me1 KO (Het) mice in male progeny. (C) Breeding scheme for paternal inheritance of the Me1 null allele. (D) Number of pups (n = 215) and corresponding genotypes compared to Mendelian ratios. No significant trends noted. (E) Breeding scheme for maternal inheritance of the Me1 null allele. (F) Number of pups (n = 121) and corresponding genotypes compared to Mendelian ratios. Non-Mendelian transmission noted in pups overall (p = 0.0451), particularly in male progeny (p = 0.0187).
Table 2.
Complete blood count values from Me1 WT and Me1 KO mice at 8 weeks of age.
Fig 3.
ME1 protein expression is notably reduced in Me1 KO mice with no impact on the NADPH/NADP+ ratio.
(A-C) Me1 WT, Het, and KO mice were examined for histology and immunohistochemistry, and representative slides were taken. (A) The liver was imaged at 10X magnification (scale bar = 400 μm) with a 1:500 ME1 antibody stain ratio for IHC. (B) The thymus at 20X magnification (scale bar = 200 μm) with a 1:250 ME1 antibody stain ratio. (C) The testes at 10X (scale bar = 400 μm) with a 1:250 ME1 antibody stain ratio. Across the three tissues, Me1 Het mice exhibited partial knockout of ME1 and Me1 KO mice exhibited total knockout. NADP+, NADPH, and NADP+/NADPH ratios in (D) liver, (E) thymus, (F) testes, (G) kidney, (H) pancreas, and (I) spleen from Me1 wild type versus Me1 null animals, as assessed by LC-MS. Males and females were analyzed in equal proportion (S2 Fig) Serum chemistry assessment checked for (J) CREA, creatinine; (K) GLUC, glucose; (L) TPRO, total protein; (M) CA, calcium; (N) ALT, alanine transferase; (O) TBIL, bilirubin; (P) BUN, blood urea nitrogen; (Q) ALKP, alkaline phosphatase; and (R) ALB, albumin. Each data point represents an individual animal. Significance was determined by student’s t-test.
Fig 4.
Me1 cKO mice behavior and physiology is not statistically different from Me1 FL/FL mice.
(A) PhenoMENA behavioral analysis measured good scoring (GS), moderate scoring (MS), and bad scoring (BS) behaviors, along with grip strength (GRiP) and explorative proclivity (GRiD). GRiP scores: normal control (FL/FL) = 0.130 ± 0.007 kg; cKO = 0.143 ± 0.007 kg. No differences between groups noted, and all values were within normal ranges for BL6 mice. (B) Accelerating rotarod performance test. Best performances (sec) were recorded for each mouse and were averaged for both groups. FL/FL = 130 ± 10 sec; cKO = 90 ± 10 sec. (C) Average heart rate. FL/FL = 510 ± 10 BPM; cKO = 510 ± 20 BPM. Average systolic blood pressure. FL/FL = 139 ± 5 mmHg; cKO = 147 ± 10 mmHg. Average mean arterial pressure. FL/FL = 114 ± 4 mmHg; cKO = 118 ± 10 mmHg. Average diastolic blood pressure. FL/FL = 102 ± 3 mmHg; cKO = 104 ± 10 mmHg. (D) Murine KPC pancreatic tumor cells orthotopically implanted in mice were weighed and compared to body weight (BW). Average tumor weight. FL/FL = 0.8 ± 0.1 g; cKO = 0.94 ± 0.05. Tumor weight / body weight ratio. FL/FL = 3.4 ± 0.3%; cKO = 4.0 ± 0.2%.
Table 3.
Complete blood count values from Me1 FL/FL and Me1 cKO mice.
Fig 5.
ME1 protein expression is notably reduced in cKO mice with no hematological phenotype.
(A-C) Me1 FL/FL and Me1 cKO mice were examined for histology, immunohistochemistry, and serum chemistry. Slides were stained with H&E and a 1:500 ME1 antibody stain ratio for IHC. Representative slides are presented. (A) The liver was imaged at 10X magnification (scale bar = 400 μm), (B) the thymus at 20X (scale bar = 200 μm), and (C) the testes at 10X (scale bar = 400 μm). Across the three tissues, Me1 KO mice exhibited significant loss of ME1. (D-L) Serum chemistry assessed for (D) CREA, creatinine; (E) GLUC, glucose; (F) TPRO, total protein; (G) CA, calcium; (H) ALT, alanine transferase; (I) TBIL, bilirubin; (J) BUN, blood urea nitrogen; (K) ALKP, alkaline phosphatase; and (L) ALB, albumin. Collection of insufficient blood volume resulted in lower sample size for TPRO and ALB.