Genetically engineered mouse models (GEMMs) have become imperative to the drug discovery process, serving as unique tools that can model specific disease pathologies and direct the development of effective and well-tolerated therapeutic strategies. Mirimus specializes in engineering customized mouse models with inducible and reversible gene silencing capabilities by harnessing the power of RNA interference and advanced genome editing technologies. These mouse models have created an unprecedented ability for the rapid identification and validation of putative drug targets as well as predicting potential toxic side effects prior to the development of drug compounds.
Suppressing gene function by RNA interference
Mirimus uses advanced RNAi technology with short hairpin RNAs (shRNAs) expressed within the context of an endogenous microRNA backbone. In comparison to siRNAs and simple stem-loop shRNAs, miRNA-based structures mimic endogenous RNAi triggers and thus engage the endogenous RNAi machinery to induce enhanced gene suppression with dramatically reduced off-target effects [Fig 1a].
Mirimus continues to work hard to bring to you the latest improvements to RNAi technology. Our optimized miRNA backbone, “miR-E”, was designed for the most efficient shRNA processing in order to produce maximal gene knockdown [Fig 1b, Fellmann reference].
Figure 1a. RNAi-mediated knockdown of gene expression can be triggered transiently by transfection of siRNAs, or stably by stem-loop structures encoded from DNA-based vectors. shRNAs based on the fold of the mir-30 microRNA act as physiologic triggers of RNAi and can work more effectively at single copy in the genome than standard stem-loop shRNAs. Dickins RA et al. (2005) Nat. Gen. An optimized miRE backbone further enhances shRNA potency at single copy integration (Fellmann reference).
Fig 1b. Comparison of konockdown efficiecy of shRNAs in miR-30 and miR-E backbones assessed by a functional reporter assay (left). Western blot analysis of Pten knockdown by miR-30 and miR-E shRNAs (right).
Mirimus employs an innovative Sensor Ping-Pong assay capable of deciphering elaborate shRNA libraries to identify the most potent shRNAs for each gene. More than 25,000 shRNAs can be screened simultaneously for functionality and accurate prediction of their silencing capacity. Our leading shRNA technology ensures potent gene suppression in vivo.
Fig 2. Large scale shRNA libraries are filtered through the Sensor assay to identify potent shRNAs (left). As a readout, a score indicates the perfomance of each shRNA in the assay (middle). Western blot analysis of Trp53 knockdown by shRNAs targeting Trp53 from a single copy genomic integration (right)
|Predicted miR-E shRNA set
(6-8 shRNAs per gene)
|Sensored shRNAs (2-5 shRNAs per gene)||1-3 wks|
|Validated shRNAs by mini reporter assay
(2-5 shRNAs per gene)