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In these triple mutant mice the absence of keto acid dehydrogenase (BCKDH) activity and E2 protein in liver tissue due to the Dbttm1Geh knock-out mutation is rescued by the two transgenes, rendering this strain as a useful model for Maple Syrup Urine Disease (MSUD).
Gregg E Homanics, University of Pittsburgh
Genetic Background | Generation |
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Allele Type |
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Transgenic (Transactivator) |
Allele Type | Gene Symbol | Gene Name |
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Targeted (Null/Knockout) | Dbt | dihydrolipoamide branched chain transacylase E2 |
Allele Type |
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Transgenic (Inducible, Hypomorph, Inserted expressed sequence, Humanized sequence) |
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.
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. These 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.
Expressed Gene | tTA, tetracycline-controlled transactivator, E. coli |
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Site of Expression | Expresses tTA at high levels in the liver; see Stock No. 003563 |
Site of Expression | The targeted mutation leads to absence of branched-chain keto acid dehydrogenase activity and E2 protein in liver tissue. |
Expressed Gene | DBT, dihydrolipoamide branched chain transacylase E2, human |
Expressed Gene | tTA, tetracycline-controlled transactivator, E. coli |
Site of Expression | Expresses tTA at high levels in the liver; see Stock No. 003563 |
Allele Name | transgene insertion 5, Hermann Bujard |
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Allele Type | Transgenic (Transactivator) |
Allele Synonym(s) | g(tTALap)5Bjd; LAP-tTA; Tg(tTALap)5Uh |
Gene Symbol and Name | Tg(Cebpb-tTA)5Bjd, transgene insertion 5, Hermann Bujard |
Gene Synonym(s) | |
Promoter | Cebpb, CCAAT/enhancer binding protein beta, rat |
Expressed Gene | tTA, tetracycline-controlled transactivator, E. coli |
Site of Expression | Expresses tTA at high levels in the liver; see Stock No. 003563 |
Strain of Origin | NMRI |
Chromosome | UN |
General Note | Transgenic mice on a C57BL/6J background are viable and fertile, and express tTA in the liver. |
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). |
Mutations Made By | Dr. Hermann Bujard, Universit¿t Heidelberg |
Allele Name | targeted mutation 1, Gregg E Homanics |
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Allele Type | Targeted (Null/Knockout) |
Allele Synonym(s) | E2 KO |
Gene Symbol and Name | Dbt, dihydrolipoamide branched chain transacylase E2 |
Gene Synonym(s) | |
Site of Expression | The targeted mutation leads to absence of branched-chain keto acid dehydrogenase activity and E2 protein in liver tissue. |
Strain of Origin | (129X1/SvJ x 129S1/Sv)F1-Kitl+ |
Chromosome | 3 |
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. |
Allele Name | transgene insertion A1, Gregg E Homanics |
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Allele Type | Transgenic (Inducible, Hypomorph, Inserted expressed sequence, Humanized sequence) |
Allele Synonym(s) | TRE-E2 (line A) |
Gene Symbol and Name | Tg(tetO-DBT)A1Geh, transgene insertion A1, Gregg E Homanics |
Gene Synonym(s) | |
Promoter | tetO, tet operator, |
Expressed Gene | DBT, dihydrolipoamide branched chain transacylase E2, human |
Expressed Gene | tTA, tetracycline-controlled transactivator, E. coli |
Site of Expression | Expresses tTA at high levels in the liver; see Stock No. 003563 |
Strain of Origin | C57BL/6J |
Chromosome | UN |
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 (Dbttm1Geh) 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 addition of the c-myc tag interferes with BCKDH subunit assembly (ie. interaction the human E2 subunit with mouse E1 and E3 complexes), thus reducing enzymatic activity. |
When maintaining a live colony, mice that are heterozygous for the targeted mutation and carriers of both transgenes can be bred together. Loss of one or both of the transgenes results in perinatal lethality (and Maple Syrup Urine Disease or MSUD phenotype) for mice homozygous for the targeted mutation.
When using the MSUD murine model mouse strain in a publication, please cite the originating article(s) and include JAX stock #006999 in your Materials and Methods section.
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