An inducible gene knock-out mouse model is defined as a model where a target gene is inactivated at a specific time point, in specific tissues. By this means, both spatial and temporal control of gene activity can be achieved. Typically, an important tool for establishing sophisticated animal models of human diseases is used for this purpose, that is the Cre/LoxP site-specific recombination system. Using the most commonly used Cre/LoxP site-specific recombination system, Creative Biolabs offers customized inducible gene knock-out services for the construction of models that can be controlled both spatially and temporally.

Mechanisms of the Inducible System

To be specific, the induction is performed based on the existence of estrogen receptor (ER), a receptor that translocates from the cytoplasm to the cell nucleus to regulate gene expression after binding to the corresponding ligand, estrogen or tamoxifen. To eliminate the effect of endogenous estrogen, the binding domain of ER is artificially mutated and therefore it is only capable of binding to exogenous estrogen analogues, tamoxifen. In this strategy, Cre is ligated to the ER and this fusion protein (e.g., CreERT2) will stay in the cytoplasm before the administration of tamoxifen, leading to functional expression of the endogenous gene. However, when tamoxifen is applied to the animal, either topically or through injection, it will bind to the ER as a ligand and thus activate the Cre-mediated recombination process. This way, the knock-out model can be obtained and timing control of inactivation can be guaranteed.

Inducible conditional knock-out by breeding with inducible tissue-specific Cre mouse. (Adams et al. 2008) Fig.1 Inducible conditional knock-out by breeding with inducible tissue-specific Cre mouse. (Adams et al. 2008)

Variations

Another inducible Cre system takes advantage of the reverse tetracycline-controlled transactivator (rtTA). Doxycycline is administered to activate the rtTA that, in turn, will induce the transcription of Cre. What's more, Cre can also be delivered through injection of a viral vector. In this case, when and where the Cre is expressed is controlled by the timing and site of injection for the virus. The amount of DNA rearrangement can be adjusted by varying the titer of the virus. These methods are less frequently used but still prove to be useful alternative ways.

Advantages and Limitations

One biggest advantage of these inducible knock-out animal models they share with any other form of conditional knock-out models is that they avoid a deadly phenotype caused by a conventional point mutation. Further, timing control of this model is another considerable strength.

However, when designing the process of creating such a model, careful considerations and a thorough analysis are necessary in order to avoid down-regulated expression of the endogenous gene or truncated protein expression. Besides, there will be a longer timeline due to an extra breeding step with a Cre mouse used to delete the target gene.

CRISPR/Cas9-Based Targeting Strategy

Creative Biolabs is on the leading edge of the genetically engineered mice model creation field. With the latest CRISPR/Cas9 technology, virtually mouse model of any genetic background can be generated, either by microinjection or via CRISPR-based technology in ES cells, to meet every specific requirement.

Meanwhile, Creative Biolabs offers other types of knock-out models that you may be interested in:

At Creative Biolabs, you will find the most comprehensive services associated with animal model creation. Models of any genetic background can be generated with our cutting-edge technology and abundant experience. If you're interested in our services, contact us for more information.

Reference

  1. Adams, D. J.; Weyden, L. V. D. Contemporary approaches for modifying the mouse genome. Physiological Genomics. 2008, 34(3):225-38.

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