Gaining Inducible Control

Inducible Control of Your Tissue-Specific Animal Model

The Cre-lox system is commonly used to provide tissue-specific gene knockout.  In order for this to happen in vivo, two mouse lines are mated together – The tissue-specific Cre line and a second mouse line containing your gene of interest flanked by loxP sites (floxed mouse). Cre recombines the loxP sites, creating a tissue-specific knockout of your gene of interest. The system is straightforward, reliable and there are many Cre lines now available to choose from for your studies. However, sometimes you require more than just tissue-specific control of your animal model for your experiments. In this blog we describe two inducible systems which add new options for your animal model by providing temporal control.

Drug-inducible Cre recombinase system

As soon as Cre is expressed in the cell, it moves into the nucleus and recombines loxP sites, creating a gene knockout. When Cre is driven by a tissue-specific promoter, knockout will occur in those specific tissue cells. However you may want timed control over your tissue-specific animal model as well in order to expand your experiments. This is possible by using an inducible system called Cre-ERT. With this system, Cre is fused to a mutated ligand-binding domain from the estrogen receptor (ER). Cre-ERT is expressed as normal but is unable to bind DNA in the nucleus because it is sequestered in the cytoplasm.  Cre becomes localized to the nucleus only when synthetic steroid, such as Tamoxifen, is present. The Cre-ERT system can be tissue-specific, just like Cre, and has inducible control because Cre-ERT waits for the drug to be administered in order to become active. Just as with the Cre-lox system, you need two lines in order to achieve knockout: The Cre-ERT mouse line and your floxed mouse line (that you can also use with standard Cre for comparison). The limitations with using Cre-ERT when compared to Cre alone are similar, e.g. you will need to demonstrate that the knockout is occurring where and when you think it is. Otherwise the systems are comparable and the only difference is the administration of the drug, which can occur by injection or ingestion. The Cre-ERT systems has been improved to make it more sensitive to the drug and in this case you may find lines called Cre-ERT2 available from collaborators or in a repository.

Inducible transgene expression with Tet-ON systems

Cre-ERT2 is a way to have inducible knockout but what if you want inducible activation of your cDNA? The Tet-ON system is used to provide inducible activation of a target cDNA in the presence of a drug such as Dox.  More specifically, the Tet-ON system utilizes a mutated transactivator called reverse tTA (rtTA). In this system the presence of Dox allows the system to be turned on and your cDNA to be expressed. Two mouse lines are required for this to work: A tissue-specific rtTA-expressing mouse line and a second mouse line with a specific DNA element (TRE) driving your cDNA.  Dox can be added to food or drinking water to activate your cDNA. Take away Dox to return to normal.

Tissue-specific control of this system can get tricky because limited tissue-specific rtTA lines exist. In order to address this, ingenious has created its Inducible Rosa-Express™ system where only two mouse lines are required and tissue specificity is provided by one of the many Cre lines available. By designing a mouse line that contains a floxed stop cassette and all of the necessary Tet-On control components, we enable tissue-specificity and inducibility of your cDNA without the need for transactivator lines. Simply mate our Inducible Rosa-Express™ line containing your specific cDNA to one of the tissue-specific Cre recombinase mouse lines that you probably already have in your mouse room and introduce Dox for inducible activation of your cDNA in your tissue of interest.

References

• Elefteriou F1, Yang X. 2011. Genetic mouse models for bone studies—Strengths and limitations. Bone 49(6):1242-54.
• The ABC’s of choosing a Cre strain to generate KO mice
• Zhong ZA1, Sun W1, Chen H1, Zhang H2, Lay YE1, Lane NE1, Yao W3. 2015. Optimizing tamoxifen-inducible Cre/loxp system to reduce tamoxifen effect on bone turnover in long bones of young mice. Bone 81:614-619.

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