RNA interference (RNAi)-mediated gene knock-down, a method of gene silencing, has developed into a fast alternative method of conventional knock-out approaches. Particularly, Creative Biolabs offers knock-down models that can be accomplished through short hairpin (sh) RNAs expressed from transgenic vectors as well as Cre recombinase or doxycycline to elicit body-wide (constitutive knock-down mice), cell type-specific, or inducible gene silencing (conditional knock-down mice).
Constitutive Knock-down
Constitutive knock-down models are animals that stably express a chosen shRNA sequence, thereby permanently silencing a target gene in vivo. The production of this type of knock-down mice is achieved via insertion of shRNA vectors into the Rosa26 locus of ES cells by recombinase-mediated cassette exchange and the subsequent generation of germ line chimeras. A single shRNA vector copy inserted within the universal docking site, Rosa26 locus, is sufficient to elicit reproducible body-wide gene silencing. Constitutive shRNA expression vectors produce body-wide gene silencing at all times in development and adulthood, however, this approach is limited by the embryonic lethality of many mutants and the lack of tissue-specificity, thus narrowing the mechanistic study of gene function.
Cre Recombinase-inducible System
Cre/loxP system is used for the conditional knock-down of gene expression in specific tissues, which can avoid the limitations of a constitutive knock-down strategy. First, a loxP-flanked transcriptional ATOP cassette is inserted into the HindIII restriction site to prevent the production of a complete shRNA. Then, by breeding it with a Cre expressing mouse, the STOP cassette is excised from the shRNA construct, allowing the transcription of a functional shRNA. As a result, this approach enables sophisticated studies of gene function by tissue- and time-specific gene silencing.
Introduction of Dox-inducible System
Based on the E. coli tetracycline operon, the tet repressor/tet operator system represents an invaluable tool for the reversible and systemic induction of shRNA expression and gene silencing. In the absence of an inducer, the tetracycline repressor (tetR) protein binds tightly to the tetracycline operator (tetO) DNA sequence and effectively inhibits the transcription of shRNA due to steric hindrance.
However, when tetracycline is present or when its derivative doxycycline tetR dissociates from tetO, the transcription is allowed to proceed. In this case, a tetO-modified H1 promoter is used, which will drive expression of shRNAs only when doxycycline is added. Consequently, the target gene can be silenced upon addition of doxycycline but is expressed at wild-type levels in the absence of inducer. More importantly, this system offers the opportunity to reverse the induced gene knock-down at a given time. This DOX-inducible system can provide unique applications to study gene function in mice that cannot be achieved with knock-out technologies.
Features of RNAi Mouse Models
Before it is injected into the ES cells, the design and construction of the shRNA vector is needed and is often a critical process in the production of knock-down models. Moreover, to achieve a satisfying level of gene silencing in vivo, it is instrumental to identify well working RNAi target sequences through initial in vitro screening. At Creative Biolabs, our scientists have accumulated abundant experiences in vector construction as well as the screening of target sequences, and thus we are confident to offer the most reliable services.
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Creative Biolabs provides state-of-the-art customized animal model creation services to clients for different in vivo study requirements. Please feel free to contact us if you are interested in any of these services.
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