Conditional Knockout with Reporter
Adding a reporter gene to a conditional knockout model enables you to visualize when and where the gene is inactivated. This gives you confirmation at the level of single cells that recombination has knocked out the target gene. Different strategies for incorporating reporters are available, depending on the genomic structure of your gene of interest.
Every experiment using a conditional knockout mouse model has two requirements: demonstration of successful knockout and labeling of affected cells and tissues. By adding a reporter to your targeting strategy both these conditions are satisfied as part of the experiment. At the same time as your gene of interest is floxed a reporter gene sequence is introduced, with expression of the reporter controlled by Cre recombinase activity. Depending on your preferred strategy the reporter can be activated or deactivated at the same time as your target gene is knocked out. This gives you cell-specific labeling of successful knockout. An important consideration is that the level of reporter gene expression depends on the expression level of your targeted gene. Besides the strategies described below, consider our No-Doubt Conditional KnockoutTM model to guarantee strong reporter expression after knockout.
Conditional Knockout With Inverted Reporter
Cre recombinase is most commonly used to conditionally delete sequences flanked by loxP sites. However, by using atypical sequences and orientation for the loxP sites it is possible to invert the flanked sequence rather than delete it. Taking advantage of this, a reporter gene can be placed next to a targeted exon, with the reporter sequence oriented in the opposite direction to the target gene. Recombinase activity inverts the region, effectively deleting the target exon by flipping its orientation. At the same time the reporter is placed in frame with the coding sequence, triggering its expression. The reporter sequence ends with a poly-A to help ensure early termination of the target gene’s transcript. Through this disruption the gene is inactivated.
An example of this complex model system has recently been published
by our client Andrea Meredith at the University of Maryland. Click here to read her published work.
Conditional Knockout With Reporter For Small Genes
For smaller genes a conditional knockout strategy may target the majority of the coding sequence. In this case a reporter gene can be placed after the last exon of the targeted gene – the endogenous poly-A of the gene will block expression of the reporter. After recombination the reporter is brought into frame and its expression is activated. Our No-Doubt Conditional KnockoutTMstrategy can also provide strong reporter expression after knockout, and can be used with genes of almost any size.
Reporter Expresses Before Cre Recombination
Another option for small genes is to tag your gene of interest with a reporter gene that is included in the floxed region and expressed along with your target gene prior to recombination. After the conditional knockout is triggered the level of fluorescence will indicate remaining protein levels.
Reporter Expresses Before and After Cre Recombination
An even more sophisticated approach is to have one reporter gene expressed together with the gene of interest, and a different reporter expressed once the gene of interest has been inactivated. The image below shows a strategy that accomplishes this. The GFP is expressed along with the gene of interest from the gene’s endogenous promoter. Due to the arrangement of the loxP sites, Cre-mediated recombination deletes major exons of the target gene and the GFP cassette. Following Cre-mediated recombination, the RFP is expressed from the endogenous promoter of the target gene, for distinguishing the null gene allele from the floxed gene allele.