RNAi mouse model engineering begins with precise genome targeting into pre-engineered mouse embryonic stem cells (ESCs). Mirimus utilizes tetracycline-responsive, fluorescence-coupled shRNA technology to enable inducible and regulatable shRNA expression in mice. Our TET-inducible shRNAs are inserted at single copy downstream of the Col1a1 locus using precision genome targeting (reference Premsrirut et al, 2011) in order to eliminate any variation in copy number integration and expression patterns often associated with transgenic mouse production. Targeted ESCs can then be used to generate transgenic animals through standard blastocyst injections with nearly 100% chimerism success rates.
Tet-inducible system for regulated gene expression
Mirimus employs TET-system technology (provide a link) to enable inducible and reversible RNAi mediated gene silencing. Two components expressed from unique genomic loci are required: TET-regulated shRNA and a reverse tet-transactivator or rtTA. In presence of a tetracycline analog such as doxycycline, rtTA binds to the TRE promoter and drives expression of the shRNA.
Fig 3. The TET-inducible system. In presence of doxycycline, rtTA binds to the TRE promoter resulting in shRNA expression and subsequent target gene silencing. In the absence of doxycycline, rtTA is unable to bind to the TRE promoter thus switching shRNA expression off and allowing endogenous gene expression to return to normal levels.
Fig: Pre-engineered KH2 ES cells harboring Rosa26-M2rtTA targeted with the indicated Tet-regulated shRNAs and treated with doxycycline to induce shRNA expression. Premsrirut PK et al. (2011) Cell.
|Embryonic Stem Cell Targeting (2 vials)||12-14 weeks|