Production and characterization of murine models of classic and intermediate maple syrup urine disease

Abstract

Background: Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model.

Methods: To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels.

Results: By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5-6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model.

Conclusion: These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease.

Figure 1

E2 gene knockout mouse production. A, Gene targeting strategy used for targeting the E2 locus in mouse ES cells. The targeting construct was designed to delete 1.67 kb of sequence between an EcoRV site in Exon 4 and a Smal site in intron 5. The wild type E2 gene contains an ~16 kb BglI restriction fragment that hybridizes to the Exon 6 specific probe. A correctly targeted E2 locus harbors an ~11 kb BglI restriction fragment that hybridizes to the same probe. Note that the probe will not detect random integration of the targeting vector because it is external to the targeting vector. B, Southern blot analysis of Bgll digested genomic DNA derived from the parental wild type ES cell line (R1), a heterozygous targeted ES cell line (362), and from wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. The blot was hybridized with an Exon 6 specific probe. C, Immunohistochemical analysis of fresh frozen liver sections from control (+/+) and E2 knockout (-/-) postnatal day 1 mouse pups. Sections were stained for E2 using an E2 specific antibody (green) and a nuclear stain (blue). Note the complete absence of E2 immunoreactivity in the section from the knockout mouse. D, Similar results were observed upon immunohistochemical analysis of primary mouse embryonic fibroblasts (MEFs). Note that the readily detectable signal for E2 in the control cells was present in a pattern characteristic of mitochondria, the subcellular location of BCKDH.

Figure 2

Biochemical characterization of the classic MSUD murine model. A, BCKDH enzyme activity in liver of newborn wild type control (+/+), heterozygous (+/-), and homozygous (-/-) knockout mice. Enzyme activity was significantly reduced in +/- liver compared to +/+, and was below the level of detection in -/- liver. B, Total BCAA concentrations in blood of mice. Total BCAA represent the sum of leucine, isoleucine, and valine. Total BCAA concentrations in blood from -/- mice were significantly elevated compared to +/+ and +/-. C, Ratio of total BCAA to alanine in blood of mice. This ratio was significantly elevated in -/- mice compared to +/+ and +/- mice. The numbers on the bars indicates the number of mice analyzed. *, Significantly different from +/+ (P < 0.001); **, significantly different from +/+ and +/- (P < 0.001).

Figure 3

Transgenic mouse production and characterization. A, Transgenic strategy used to produce mice that express human E2. LAP-tTA transgenic mice have been previously described [34]. These mice express the tetracycline-controlled transactivator (tTA) from the liver specific LAP promoter. The TRE-E2 transgene contains the tetracycline response element (TRE) as part of the promoter, a synthetic intron (thin line), the human E₂ cDNA, an alanine spacer, a c-myc epitope tag, and SV40 derived polyadenylation sequence. This construct was used to create several lines of transgenic mice. B, Western blot analysis of E2 protein in liver of control and intermediate MSUD mice. Note that the amount of human E2 protein (predicted MW ~54 Kd) in mice from Lines A and 525 A was variable but in many of the animals the amount was similar to the amount of mouse E2 (MW=~47 Kd) in control animals. Re-probing with a c-myc tag antibody confirmed the presence of the transgene derived, c-myc tagged, human E2 in transgenic mice but not in controls. Western blot analysis of brain (C), kidney (D), and muscle (E) revealed negligible amounts of transgene derived E2 in those tissues. All blots were re-probed with an actin antibody to allow amount of protein loaded in each lane to be compared.

Figure 4

Characterization of the intermediate MSUD murine model. A, Survival analysis of transgenic rescue lines. Presented are percentages of mice alive at weaning from each transgenic line tested that had the rescue genotype (i.e., homozygous for knockout of endogenous E2 and positive for both the LAP-tTA and TRE-E2 transgenes). Also plotted is the theoretical maximum frequency at which this genotype is expected in this population of animals. The numbers on the bars indicates the numbers of observations for each line. B, Ratio of total BCAA to alanine in blood of mice from controls and Lines A and 525A. Total BCAA represent the sum of leucine, isoleucine, and valine. BCAA/alanine values were significantly greater for Lines A and 525A compared to controls at all ages tested. The numbers on the bars indicates the numbers of samples analyzed. *, P ≤ 0.01; **, P < 0.005. C, BCKDH enzyme activity in liver of control and Lines A and 525A mice. *, P ≤ 0.001; **, P ≤ 0.0001. D, Survival curves for mice from Lines A and 525A. Rescue mice alive at weaning were monitored until they were moribund and subsequently sacrificed or were found dead in their cages.