How to Obtain a Conditional Knockout

Floxed Mice and the Cre-Lox Procedure

Conditional knockouts are firmly established as indispensable tools for researchers using mouse models. This model type has aided in studies across an incredible range of topics, from rare genetic diseases to fundamental biological processes. By combining tried-and-true methods with newly-developed techniques this type of mouse model will continue to be valuable for basic and applied research for the foreseeable future.

The value of conditional knockout mice is clearer with an understanding of the components that go into making and using the models. Conditional knockout mice are used as part of a system that’s commonly referred to as “Cre-lox” which names the two elements that make it work. “Cre” is Cre recombinase, a DNA modifying enzyme originally derived from a virus. This enzyme seeks out DNA with a specific sequence known as a “lox” site. When two lox sites are close together the Cre enzyme can trigger a dramatic change in the DNA molecule. This reaction can be controlled by making small changes in the DNA sequence of the lox sites, so the Cre-lox system can be used to alter DNA in multiple ways. In the case of conditional knockout mouse models the goal is to have a specific gene function normally at first, then have Cre recombinase inactivate the gene at the right time.

Using the Cre-lox system for conditional knockout requires the creation of at least one genetically modified mouse line. Two lox sequences must be carefully inserted into precise locations to create a “floxed” gene. Ideally the floxed gene will not be disrupted at all by the insertion of the lox sequences – they are placed in locations where a small change doesn’t have an effect. If no Cre recombinase is added then the floxed gene should function as normal and the mice should be healthy. However when Cre is added the power of the conditional knockout is revealed: Cre triggers a change in the DNA sequence that inactivates the floxed gene. This enables experiments to determine the function of the gene and understand what happens when it is inactivated. Furthermore a great degree of control is possible over Cre so a wide variety of experiments are possible.

Conditional knockout mice have that name because the floxed gene can be knocked out specifically under certain conditions and this control is possible because Cre can be regulated in multiple ways. Often the genetically modified mouse line with a floxed gene is crossed to another line that expresses Cre only in certain cells in the animal, for example only in one organ. Cre triggers DNA changes that inactivate the floxed gene and since Cre is only active in certain cells the gene is conditionally knocked out in just those cells. Additional levels of control are possible by altering the Cre enzyme itself. One example is a modified Cre that only triggers DNA changes when a certain drug is added. With that additional level of control the floxed gene can be knocked out in certain cells and also the timing of knockout can be chosen. This level of control may be needed if the gene of interest is required for embryonic development for example. With genes like that an early knockout results in pups never being born, so precise control is required to make experiments possible.

Every experiment using Cre-lox for conditional knockout must account for specific features of the floxed gene as well as regulation of Cre. Introducing lox sites into the genome should not affect the gene of interest and this must be carefully checked. Likewise Cre activity must be verified, for example to confirm that Cre is only active in the desired cells. With careful planning before any new mouse models are generated the conditional knockout strategy can make exciting new discoveries possible.

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