B6.Cg-Gt(ROSA)26Sor^{tm37(H1/tetO-RNAi:Tafazzin)Arte}/ZkhuJ

Stock number: 014648
Product name: TAZKD
Research areas: Cardiovascular Research, Metabolism Research, Research Tools

Description

This knock-in mutation (also referred to as a tafazzin knock-down or TAZKD) strain is a tetracycline inducible shRNA-mediated TAZ knock-down mouse model of Barth syndrome, and may also be useful in studies of lipid metabolism, and myocardial and mitochondrial physiology.

Detailed description

These mutant mice have a tetracycline inducible Taz specific short hair pin RNA (shRNA) driven by the endogenous mouse Gt(ROSA)26Sor promoter. Expression of the shRNA is controlled by the transcription of the H1 RNA polymerase III promoter, which is coupled to a tet-operator (tetO) sequence. Expression of the shRNA is blocked by codon-optimized version of the tet repressor itetR, which is part of the allelic construct found in this mouse. Doxycycline (dox--a tetracycline analog) treatment decreases the affinity of the itetR for the tetO sequence, allowing transcription of the shRNA.

Dox-induced Taz gene silencing is detected in heart (to ~3.7% of wildtype), skeletal muscle (to ~11.2%), liver (to ~8.9%) and brain (to ~3.4%) by RT-PCR analysis. Gene product (mRNA) in transgenic mice without dox induction is reduced by 35% of wildtype control levels. Withdrawal of dox at 4 weeks partially reverses the reduction of Taz expression. Protein product is reduced in cardiac (by 97%) and in skeletal muscles (by 86%) as detected by Western Blot analysis. Dox treated mutants, at 8 months of age, weigh 17% less than controls and exhibit left ventricular cardiac dilation and mass reduction. In dox-treated 2 month old mutants, levels of tetralinoleoyl cardiolipin in cardiac and skeletal muscles are reduced, with a shift to more saturated cardiolipins and an accumlation of monolysocardiolipin (MLCL) resulting in an increase in the monolysocardiolipin/cardiolipin ratio. Ultrastructural mitochondrial and sarcomeric abnormalities are observed in dox-treated 8 month old mutants, including mitochondrial aggregations, mitochondrial vacuoles, disorganized myofibrils and endoplasmic-reticular membranes in skeletal muscle and swollen mitochondria and reduced myofiber density in cardiac tissue. Skeletal muscle contraction is impaired. Mice that are heterozygous for the targeted mutation are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities in the absence of tetracycline or any similar analog. The Donating Investigator reports that although homozygotes are viable, in the absence of dox do not live long.

Development

A targeting vector containing Taz specific shRNA under the control of the H1 of RNA polymerase III promoter and a tetracycline operator (tetO), a neomycin selection cassette, and a constitutive expression cassette encoding the codon optimized version of the tet repressor: CAGGS-itetR, was inserted into the Gt(ROSA)26Sor locus by recombinase mediated cassette exchange (RMCE). Transcription of the H1 RNA polymerase III promoter is blocked in cells expressing itetR. The construct was electroporated into 129S6 derived ES cells. Correctly targeted ES cells were injected into tetraploid blastocysts from B6D2F1 mice. The donating investigator reported that the mice were backcrossed to C57BL/6J mice for 5 or 6 generations (see SNP note below) prior to sending to The Jackson Laboratory Repository. Upon arrival, heterozygous sperm was frozen. To generate the live colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

In 2021, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Four of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm37(H1/tetO-RNAi:Tafazzin)Arte}
Name: targeted mutation 37, TaconicArtemis
MGI accession ID: MGI:4887237
Generation method: Targeted
Attributes: Inducible; Knockdown
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: C57BL/6
Molecular note: A short hairpin RNA directed against Taz was targeted into the Rosa26 locus. The shRNA sequence is under the regulation of the H1/tetO operator fusion promoter. Correct mRNA knock-down was confirmed in embryonic stem cells.