STOCK Dbt^tm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J

Stock number: 006999
Product name: MSUD murine model
Research areas: Metabolism Research, Research Tools

Description

In these triple mutant mice the absence of keto acid dehydrogenase (BCKDH) activity and E2 protein in liver tissue due to the Dbt^tm1Geh knock-out mutation is rescued by the two transgenes, rendering this strain as a useful model for Maple Syrup Urine Disease (MSUD).

Detailed description

Mice homozygous for the Dbt (E2) targeted mutation and carrying both the LAP-tTA and TRE-E2 transgenes are viable and fertile. The E2-targeted mutation leads to absence of branched-chain keto acid dehydrogenase (BCKDH) activity and E2 protein in liver tissue, but this absence is rescued by the two transgenes: liver-directed expression of the modified human BCKDH E2 subunit from the complimentary "Tet-off" transgenes abrogates the severity of Maple Syrup Urine Disease (MSUD) phenotype observed in E2-deficient single mutant mice. Triple mutant mice are a model for intermediate MSUD (iMSUD); BCKDH activity is only 5-6% of that found in wildtype mice. This low level of BCKDH activity is sufficient to allow survival, but insufficient to normalize circulating branched chain amino acids levels. Because these mice have near normal amounts of E2 protein, but only 5-6% of normal BCKDH enzyme activity, it is probable that the c-myc tag at the carboxy-terminus of the human E2 transgene interferes with enzymatic activity. The donating investigator reports that addition of the tetracycline analog doxycycline does not affect MSUD phenotype rescue. These mutant mice may be useful in studying metabolic disease, biochemistry, and both the complete/classic and intermediate forms of Maple Syrup Urine Disease.

Development

The mutant allele of branched-chain keto acid dehydrogenase E2 subunit (Dbt gene) was created by Dr. Gregg Homanics (University of Pittsburgh) using a targeting vector designed to replace part of exon 4 and all of exon 5 with a PGKneo cassette. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were microinjected into C57BL/6J blastocysts. Chimeric mice were bred to C57BL/6J to generate heterozygous mice. Heterozygotes were then bred to LAP-tTA transgenic mice on a mixed (NMRI outbred;FVB;C57BL/6J) background. The LAP-tTA mice were created by Dr. Hermann Bujard (Universitat Heidelberg) to express tetracycline-controlled transactivator (tTA) under control of the rat liver-enriched activator protein promoter. The TRE-E2 (or tetO-DBT*) transgene, also created by Dr. Gregg Homanics, contains a tetracycline response element and minimal human CMV promoter controlling a human E2 subunit (DBT gene) cDNA which has been modified to contain a 4x alanine linker followed by a c-myc epitope tag at the carboxy terminus. After injection of the transgene into C57BL/6J embryos, founder line A mice were bred with E2-deficient, LAP-tTA double mutant mice to generate this triple mutant strain.

Allele

Symbol: Tg(Cebpb-tTA)5Bjd
Name: transgene insertion 5, Hermann Bujard
MGI accession ID: MGI:3056818
Generation method: Transgenic
Attributes: Transactivator
Marker symbol: Tg(Cebpb-tTA)5Bjd
Marker name: transgene insertion 5, Hermann Bujard
Chromosome: UN
Strain of origin: NMRI
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli
Site of expression: Expresses tTA at high levels in the liver; see Stock No. 003563
Molecular note: This transgene contains the gene encoding the tetracycline regulated transactivator protein (tTA) driven by the rat Cebpb promoter (previously called liver-enriched activator protein).
General note: Transgenic mice on a C57BL/6J background are viable and fertile, and express tTA in the liver.

Allele

Symbol: Tg(tetO-DBT)A1Geh
Name: transgene insertion A1, Gregg E Homanics
MGI accession ID: MGI:3704903
Generation method: Transgenic
Attributes: Inducible; Hypomorph; Inserted expressed sequence; Humanized sequence; Epitope tag
Marker symbol: Tg(tetO-DBT)A1Geh
Marker name: transgene insertion A1, Gregg E Homanics
Chromosome: UN
Strain of origin: C57BL/6J
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli; DBT,dihydrolipoamide branched chain transacylase E2,human
Site of expression: Expresses tTA at high levels in the liver; see Stock No. 003563
Molecular note: The TRE-E2 (or tetO-DBT) transgene contains a tetracycline response element and minimal human CMV promoter controlling a human E2 subunit (DBT gene) cDNA which has been modified to contain a 4x alanine linker followed by a c-myc epitope tag at the carboxy terminus to facilitate detection. Levels of the human E2 protein are approximately equal to mouse E2 protein in livers of control mice. However, when crossed to animals that express a transgene encoding the tetracycline-regulated transactivator protein (Tg(tTALap)5Bjd) and are homozygous for a knockout allele of mouse E2 (Dbt^tm1Geh) are treated with doxycycline, the E2 protein levels correlate with only ~5-6% of normal branched-chain keto acid dehydrogenase (BCKDH) activity. It is one hypothesis that the addition of the c-myc tag interferes with BCKDH subunit assembly (ie. interaction of the human E2 subunit with mouse E1 and E3 complexes), thus reducing enzymatic activity.

Allele

Symbol: Dbt^tm1Geh
Name: targeted mutation 1, Gregg E Homanics
MGI accession ID: MGI:3704091
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Dbt
Marker name: dihydrolipoamide branched chain transacylase E2
Chromosome: 3
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Site of expression: The targeted mutation leads to absence of branched-chain keto acid dehydrogenase activity and E2 protein in liver tissue.
Molecular note: A targeting vector was used to replace part of exon 4 and all of exon 5 with a PGKneo cassette. Southern blot and immunohistochemical analyses verify the absence of wild-type E2 transcripts and lack of E2 protein detection in homozygotes.