Knockout mice expressing a human E2 transgene model a variant form of MSUD that mimics intermediate MSUD
To create a murine model of the intermediate variant form of MSUD, we used a transgenic strategy to rescue the severe elevation of BCAA and neonatal lethality that occurs in the classic MSUD mouse model. Our strategy was composed of a two part transgenic system to express human E2 in liver on the E2 knockout background. This bi-transgenic system consisted of a LAP-tTA transgene and a TRE-E2 transgene (Fig. 3A). The LAP-tTA transgene [34] directs high levels of liver specific expression of the tetracycline-controlled transactivator (tTA), a transcription factor that stimulates expression of promoters that harbor a transactivator response element (TRE). The TRE-E2 transgene was designed to express a human E2 cDNA from a TRE containing minimal promoter upon stimulation by tTA.
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. LAP-tTA mice were previously produced and characterized by the Bujard laboratory [34]. Pronuclear microinjection was used to produce transgenic mice that harbored the TRE-E2 transgene. From injections at the University of Pittsburgh, 2 transgene positive founders were identified that led to the generation of 3 different transgenic lines (Lines A, B, & D). From the injections at the University of Cincinnati, we obtained 8 transgene positive founders. Subsequent breeding of these founders revealed that many of the animals had multiple transgene insertion sites that segregated. We established a total of 15 different transgenic lines from these founders. Limited resources allowed us to only focus on a total of 9 of these lines. These lines differed substantially in transgene copy number as compared by Southern blot analysis of tail DNA (data not shown).
Transgenic TRE-E2 mice from each line (either founders or F1 offspring) were crossed to mice that were positive for the LAP-tTA transgene and were heterozygous for the E2 knockout. Ultimately, breeding pairs were used in which the mice were heterozygous for the LAP-tTA transgene, the TRE-E2 transgene, and the E2 knockout. To efficiently screen for the ability of the transgenes to rescue the knockout from neonatal lethality, we genotyped litters at weaning. This breeding strategy is expected to result in a theoretical maximum of animals with the rescue genotype (i.e., homozygous E2 knockout and positive for both transgenes) of 14%. Fig. 4A shows the percentage of mice alive at weaning with the rescue genotype from each transgenic line tested. Considerable variability was observed between lines. The line with the highest percentage of rescue mice at weaning was line 525 A. Approximately 10% of weaned, surviving pups from this line had the rescue genotype. Thus, the 525A transgene appears to be highly effective at rescuing the neonatal lethal phenotype of the E2 knockout. In contrast, line 520B completely failed to rescue the neonatal lethal phenotype. All other lines tested produced surviving rescue animals at a frequency between 1 and 5% of weaned pups.
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. Lines A and 525A were selected for detailed characterization. Fig. 4B shows the results of blood amino acid analysis presented as a ratio of total BCAA to alanine. The BCAA/alanine values for mice from both lines were significantly elevated compared to controls for all of the time points analyzed. Note that the BCAA/alanine values for the transgenic mice are intermediate between controls and knockouts (see Figure 2C). Blood levels of alanine, glutamate, and glutamine were reduced in Line A mice, as was glutamate in Line 525A mice, compared to controls (Table 2).
Table 2  Summary of additional blood amino acid levels in the intermediate MSUD model and littermate controls as determined by tandem mass spectrometry. Samples were collected from mice that were 4–6 weeks of age. All values are mean +/- SEM.
MSUD Genotype  Alanine  Glutamate  Glutamine
(μmole/L)
control  225.2 ± 36.6 (11)  106.5 ± 10.5 (11)  67.8 ± 7.6 (11)
525A  175.0 ± 28.7 (10)  68.4 ± 7.7** (10)  58.0 ± 7.0 (10)
A  144.1 ± 16.9* (10)  65.9 ± 8.9** (10)  37.7 ± 6.5**,*** (10)
*: Significantly different from control (P < 0.05).
**: Significantly different from control (P < 0.01).
***: Significantly different from Line 525A (P < 0.05). Because the LAP-tTA mice that were used to drive expression of transgenic human E2 have been demonstrated to produce liver specific expression [34], BCKDH enzyme activity and production of human E2 protein in liver of Lines A and 525A was examined. BCKDH enzymatic activity in liver from Lines A and 525A was ~6 and 5%, respectively, of the enzymatic activity present in control liver (Fig. 4C). As shown in Fig. 3B, the amount of human E2 (predicted MW~54 Kd) in these transgenic mice was quite variable between mice. In some of these transgenic mice, the level of human E2 was approximately equal to the amount of mouse E2 (~47 Kd) produced in liver of nontransgenic control mice. The observations that these near normal amounts of E2 protein result in only ~5–6% of normal BCKDH enzyme activity suggest that transgene derived E2 is functioning at a suboptimal level. It is probable that the c-myc tag at the carboxy terminus of the transgene derived human E2 interfered with enzymatic activity. This interpretation is consistent with previous studies which have revealed that the carboxy terminus of an analogous subunit of the pyruvate dehydrogenase complex is essential for subunit interactions [35] and insertion of a Hisx6 tag on the carboxy terminus of an analogous E. coli subunit interfered with normal subunit assembly [36]. It is also possible that human E2 was not fully functional when complexed with mouse E1 and E3 subunits. Human E2 shares ~88% identity to mouse E2 at the amino acid level. We also used western blot analysis to analyze expression of the E2 transgene in brain, kidney and muscle. As shown in Figure 3C, D and 3E respectively, expression of E2 is negligible in those tissues.
Long-term survival of the mice from these two transgenic lines that survived beyond weaning is plotted in Fig. 4D. Although survival data for control animals was not collected, it is readily apparent that survival of the rescue mice was compromised. By 16 weeks, all mice of Line A were moribund and humanely sacrificed, or were found dead in their cage. Survival of Line 525A appeared to be somewhat better. At 20 weeks of age, ~12% of mice (2 of 16) were still alive. These two rare survivors died at 40 and 60 weeks of age.
In summary, these surviving mice had BCAA/alanine ratios that were intermediate between controls and knockouts and they expressed ~5–6% of normal BCKDH enzyme activity in the liver. These phenotypic observations are remarkably similar to the clinical phenotype observed in MSUD patients with the intermediate form of the disease [1]. Thus, these rescue mice represent a very useful model of the intermediate MSUD phenotype.