Methods
All studies involving animals were reviewed and approved by the University of Pittsburgh's Institutional Animal Care and Use Committee.

Gene knockout production
A genomic DNA subclone of Strain 129/SvJ DNA that contained a portion of Exons 4 and 5 of the E2 gene and flanking DNA was obtained from a P1 phage library from Genome Systems, Inc., (St. Louis, MO; '3-Hit Mouse ES Library'). A positive/negative replacement type gene targeting vector [21] was created by replacing a 1.67 kb EcoRV-Smal fragment that included a portion of Exon 4 and all of Exon 5 with the PGKneo marker gene (See Fig. 1A) from the pPNT vector [22]. The targeting construct was linearized with NotI and electroporated into R1 ES cells [23] under conditions described previously [24]. ES cells were selected with G418 (300 μg/ml; Life Technologies Inc., Gaithersburg, MD) and gancyclovir (2 μM; gift of Syntex, Palo Alto, CA). Doubly resistant clones were screened for gene targeting by Southern blot analysis of BglI digested genomic DNA. Blots were hybridized with an Exon 6 specific probe that was external to the gene targeting construct. Correctly targeted ES cells were injected into C57BL/6J blastocysts to produce chimeric mice using standard procedures. Heterozygous offspring from germline competent chimeras were intercrossed to produce wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. At all generations, +/- breeding pairs were used. Results presented here are from mice derived from the F2+ generations. All animals were of a mixed C57BL/6J × Strain 129Sv/SvJ genetic background.
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.

Production of transgenic mice
Standard molecular techniques were used to assemble the transgenic construct, pTRE-E2. This transgene contains the tetracycline responsive hCMV*-1 promoter [consisting of the tetracycline responsive element (TRE) and a minimal hCMV promoter] [25] from pTRE2 (Clontech Inc., Mt. View, CA), a chimeric intron from pCI (Promega) to increase message stability and expression [26,27], a Kozak consensus sequence at the initiation codon to optimize translation [28], the human E2 cDNA which has been modified to contain a 4× alanine linker followed by a c-myc epitope tag at the carboxy terminus to facilitate detection, and an SV40 late polyadenylation sequence from pCI for enhanced mRNA stability and translation.
The 2.48 kb TRE-E2 transgene was purified from vector sequences following digestion with XhoI and BamHI and injected into C57BL/6J or C57BL/6J × Strain 129SvEv mouse embryos at the transgenic core facilities of the University of Pittsburgh and the University of Cincinnati, respectively. Genomic DNA from the tail of mice was screened by Southern blot analysis following digestion with EcoRI and hybridization to an ~400 bp probe derived from the SV40 portion of the TRE-E2 transgene.

Production of intermediate MSUD murine model
The various TRE-E2 transgenic lines produced were mated independently to mice that were heterozygous for both the E2 knockout and the LAP-tTA transgene [Tg(tTALap)Bjd/J; Stock 3272; The Jackson Laboratory, Bar Harbor, ME; NMRI × FVB × C57BL/6J background]. Interbreeding of animals that were heterozygous for both transgenes and the knockout resulted in the production of mice with a variety of genotypes including some animals that were homozygous for the knockout and were positive for both transgenes (we refer to this genotype as the "rescue" genotype). If our strategy for rescuing the neonatal lethal phenotype of the knockout were successful, then those homozygous knockout animals that had both transgenes would survive beyond the neonatal period.

Genotyping
All mice were genotyped by Southern blot analysis. Genomic DNAs prepared from tail snips were digested with an appropriate restriction enzyme, size fractionated by agarose gel electrophoresis, blotted to nylon, and probed using standard procedures.

Immunohistochemistry
Primary mouse embryonic fibroblasts were prepared from embryonic day ~16.5–18.5 fetuses as described [29]. Fibroblasts were passed onto glass, fixed in 2% paraformaldehyde in PBS for 10 minutes, permeabilized in 2% paraformaldehyde containing 0.1% Triton X100 for 10 minutes and washed three times in PBS containing 0.5% BSA and 0.15% glycine, pH 7.4 (Buffer A). Following a 30 min incubation with purified goat IgG (50 (μg/ml) at 25°C and three additional washes with Buffer A, cells were incubated for 60 min with E2-specific antiserum [30] at 1 μg/ml followed by three washes in Buffer A and 60 minute incubation in fluorescently labeled second antibody (1–2 μg/ml). The cells were then washed six times (5 min/wash) in Buffer A and then mounted in gelvatol (Monsanto, St Louis). When livers from newborn pups were examined, fixation was by immersion in 2% paraformaldehyde followed by cryoprotection and shock freezing in liquid nitrogen cooled isopentane and sectioning (5 microns). Otherwise processing was as for the cells above (without the fixation and permabilization steps).

Amino acid analysis
Blood was collected from the retroorbital sinus or tail vein of mice and spotted on a filter paper routinely used for blood amino acid analysis for prenatal screening. Concentrations of BCAA and other amino acids in blood were determined by tandem mass spectrometry (Pediatrix Screening, Bridgeville, PA).

Assay of BCKDH activity
Livers were removed, frozen in liquid nitrogen, and stored at -80°C. At the time of enzyme assay, livers were thawed, and homogenized (1:9, w/v) in 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4. Liver homogenates were centrifuged at 600 × g for 10 min at 4°C and the supernatant fraction was saved to determine the BCKDH activity. The use of tissue homogenates was necessitated by the limited availability of liver tissue, particularly from newborn pups. BCKDH activity was determined by measuring the release of 14CO2 from α-keto [1-14C] isocaproate as described previously [31]. The complete reaction mixture contained (final volume 1 ml) 30 mM potassium phosphate buffer, pH 7.4, 0.20 mM α-ketoisocaproate, 0.40 mM CoASH, 0.40 mM thiamin pyrophosphate, 2 mM NAD+, 2 mM dithiothreitol, 5 mM Mg2+, approximately 250,000 DPM of α-keto [1-14C] isocaproate, and 0.10 ml of liver homogenate (2–3 mg protein). Assays were carried out for 15 min at 37°C, 14CO2 was trapped in hydroxide of Hyamine, and radioactivity was determined by liquid scintillation spectrometry.

Western blot analysis
Protein extracts were isolated from homogenized liver (freshly harvested and flash frozen) of wildtype, Line 525A, and Line A transgenic mice. Protein (25 μg per sample) was analyzed by electrophoresis on a 10% SDS-PAGE Ready Gel (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (Sequi-Blot; Bio-Rad) via electroblotting. All blots were probed for E2 protein using polyclonal rabbit E2 antisera (1:5,000), which detects both mouse (~47 kD) and human (~54 kD) E2 subunits ([30]; gift from Dr. Susan Hutson, Wake Forest University). Blots were re-probed with a rabbit anti-c-myc tag antibody (1:10,000; abCam, Cambridge, MA; cat.# ab9106-100). Blots were also re-probed with an antibody for β-actin (43 kD; 1:10,000; abCam; cat.# ab8227-50) to allow for loading comparisons. A goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000; Novus, Littleton, CO; cat.# NB730-H) was used for detection using the Western Lightning chemiluminescence reagent (Perkin Elmer, Boston, MA) and exposed to X-ray film.

Statistical analyses
All BCAA and BCKDH enzyme activity data are presented as the mean +/- the standard error of the mean (S.E.M.). Differences between genotypes were compared by Student's t test[32].