The basics of conditional knockout mice are familiar to any researcher who uses mouse models, even if this model type isn’t yet a part of your research. Using the Cre-lox system in an experiment seems simple: two lines are crossed, one with tissue-specific expression of Cre and one with a floxed target gene. Ideally your gene of interest is knocked out in the tissue you’re studying. Some aspects of the Cre-lox system are overlooked even by experienced researchers, and a greater understanding may alter your future experimental plans.
A conditional knockout model offers flexibility that a constitutive or conventional knockout does not, but depending on your project both model types may have applications. It’s straightforward to start with a conditional knockout model and generate a new constitutive knockout line from it, while still separately maintaining the conditional knockout line. Simply mating the conditional knockout line with a line that expresses Cre in germ cells will result in embryos carrying the deletion. This gene-inactivating mutation will be passed on, creating a constitutive knockout line for the gene.
The mechanism of Cre recombination requires two lox sites be brought together by looping the intervening DNA. In conditional knockout models the looped sequence is cut away to create a circular DNA molecule. Varying the orientation of the lox elements can cause an alternate reaction during recombination such that the lox-flanked region is inverted rather than deleted. If two wild-type loxP sequences were used to accomplish this the region would invert over and over with no control over the final orientation. It was found that alternate lox sequences could be used to create an irreversible, or locked, inversion. This can be used to place an inverted sequence in a gene where it will initially be spliced over, then after Cre inverts the region the element comes into frame, potentially serving as a reporter or modifier of the targeted gene.
The lox element used in the Cre-lox system comprises only a 34bp sequence. In most conditional knockout Cre-lox models two loxP sites are placed in introns, flanking one or a few exons. The generally low conservation of intronic sequence might lead one to believe that lox sites can be placed freely. However even such a small introduced sequence can disrupt the gene’s function by interfering with its promoter or splicing. Often the first intron of a gene will contain regulatory DNA sequences and disrupting a key transcription factor binding site could affect the expression of the gene. lox sites should be placed outside of known regulatory regions whenever possible, ideally in regions known to have low conservation across species. lox sites should also be placed at least 150 bases away from splice sites to minimize the chance of disrupting proper splicing.
Your experiments may exclusively employ well-validated Cre lines that are backed by years of successful use. However, even the most accepted Cre lines can have recombinase activity in tissues you don’t expect. The creators of your Cre line had certain experiments planned for it which were probably limited to certain conditions during one part of the mouse’s life cycle. Every way that your experiments vary from theirs creates a possibility of different Cre expression patterns. For example, the promoter driving Cre may be activated by infection or hypoxia or age, and therefore Cre could be expressed when you don’t intend it to be. While that result may be exciting to discover it’s important that it not take you by surprise. Any time a new combination of Cre line and conditional knockout line is used it’s vital to demonstrate that unexpected knockout is not occurring and disrupting your interpretation of results.
Every conditional knockout model has a unique characteristic: the chromosomal location of the targeted gene. Because the epigenetic landscape varies depending on position two conditional lines may be affected differently when used with the same Cre line. In addition, recombination frequency is affected by the distance between lox sites – greater distance results in lower efficiency. These characteristics introduce additional complexity when comparing results from different conditional knockout lines. For example, you may be studying two closely related genes where both are targeted by floxing the third exon, and you cross the two lines to the same Cre line for experiments. Although the distance between lox sites may be almost identical the epigenetic landscape could alter recombination frequency. As a result one gene may be knocked out more efficiently and if this difference isn’t recognized it can affect your experiment in an unanticipated manner. This can also affect how you validate Cre lines because a generic reporter line won’t have its construct in the same location as your target gene. Ideally your conditional knockout line will incorporate its own reporter to give a true representation of knockout efficiency.
As mentioned above, each conditional knockout line has unique features that affect the efficiency of deletion. The other variable in an experiment is the level and duration of Cre expression. It can happen that a Cre line which has reliably worked for years suddenly seems weak when you cross it with a new conditional knockout line. One possible explanation is that the Cre line was weaker than you thought, but you were using it with conditional knockout lines where the gene deletion was particularly efficient. If your Cre mice lines express in multiple tissues it’s almost certain that their expression levels will vary by cell type. Using a binary off/on reporter to determine where Cre is expressed can obscure these differences – low Cre levels might be enough to trigger the reporter but not reliably work with a conditional knockout line. A new conditional knockout line with a conditional reporter is one way to conclusively know that your target gene has been disrupted when you think it has. You will likely observe that knockout efficiency varies between tissues because of different Cre activity levels.
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