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.
