Conditional Knockout Mouse Models
A conditional knockout mouse model enables the inactivation of a gene of interest in a specific tissue and/or at a specific time point (inducible), while in all other tissues the gene should retain its wildtype function. Most commonly, the Cre-lox system is utilized for conditional deletions or mutations of a gene. Using a conditional knockout approach limits the risk of embryonic lethality as posed by conventional knockout strategies, and it opens up significantly more options as to where and when the gene can be inactivated and/or reactivated, all based on the hundreds of Cre mouse lines available. Thus, conditional knockouts are a versatile tool that can be studied across different research fields.
Advances in genetic engineering have resulted in the availability of different gene targeting options; from the traditional floxing of a specific target region, to adding reporters and floxed stop cassettes, to inducible knockout strategies. At ingenious, we will first evaluate potential design strategies for your gene of interest based on your requirements. Together we can decide which technology will achieve the best results. For your custom project, we can use CRISPR or gene targeting in ES cells, either alone or in combination, to guarantee the most reliable production of your germline confirmed mouse model. Below we review the different conditional knockout design options that are available at ingenious:
A conditional knockout mouse model enables the inactivation of a gene of interest in a specific tissue and/or at a specific time point (inducible), while in all other tissues the gene should retain its wildtype function. The Cre-lox system  is most commonly utilized for conditional mutation of a gene. The Cre recombinase enzyme interacts with short DNA sequences called loxP sites that can be engineered into the genome. When two continuous loxP sites are in the same orientation, the sequence between the loxP sites is deleted via Cre-mediated recombination. Thus, a conditional knockout allele of a gene can be engineered by flanking a critical region of the gene, such as an exon, with loxP sites. A gene engineered with such loxP sites is thus also referred to as “floxed”. For deletion of the floxed region to occur, the mouse carrying the floxed allele is typically mated with a Cre transgenic mouse, such as one that expresses Cre recombinase in the tissues or cell types of interest. A large number of tissue-specific Cre transgenic mouse lines are maintained in various repositories. For inducible, temporal control of a conditional knockout mouse model, Cre-ERT2 transgenic mouse lines are available, which allow for time point specific inactivation of a gene of interest upon administration of Tamoxifen.
Conditional knockouts have mostly replaced conventional knockouts nowadays, for avoiding potential embryonic lethality or other phenotypic complications that might result from a constitutive deletion of the gene of interest. In addition, Cre-lox conditional designs offer researchers much more versatile mouse models. The primary floxed mouse line can be mated to multiple Cre or Cre-ERT2 lines as desired. Global or ubiquitous Cre expressing lines will still allow for producing a total body knockout of the targeted gene.
General Strategy Considerations
A specific target sequence within a gene of interest is flanked by loxP sites to facilitate Cre recombinase-mediated excision, for tissue specific or temporal gene inactivation.
The following general considerations apply for conditional knockout mouse models and are evaluated by our experts for each conditional knockout model generated at ingenious.
Preservation of wildtype expression before Cre recombination
Avoid insertion of exogenous sequences into regulatory regions.
Avoid disruption of promoter elements.
Avoid disruption of splice sites.
Insert genetic elements into large introns.
We use bioinformatic and prediction tools to identify these sequences if they have been characterized at the time of target gene analysis. These findings are discussed with the client before proceeding with construction of the targeting vector.
Inactivation of the gene after Cre recombination
Target the earliest exon that will result in a frameshift and premature stop codons upon deletion.
Target an important domain.
Delete as much coding sequence as possible.
If a conventional knockout model of the gene already exists which shows no protein expression, then floxing the same region may be recommended.
The schematic below illustrates the targeting strategy of a conditional knockout mouse model in which exon 2 was flanked with loxP sites, and the Neo cassette was introduced into the intron downstream of exon 2. Upon FLP and Cre recombination, exon 2 is eliminated, and a loxP/FRT footprint remains.
The deletion of exon 2 results in a frame shift and early stop codons which can be visualized at the protein level.
Wildtype protein sequence as encoded by exon 1-4:
Protein sequence after deletion of exon 2 (as encoded by exons 1, 3 and 4):
(Frameshift mutation and predicted stop codons denoted by asterisks and yellow highlighting)
Adding a reporter gene to a conditional knockout model enables the researcher to visualize when and where the gene is expressed, or it can help to show when the gene has been inactivated. There are several strategies that can be considered, depending on the gene’s structure and the goal of the research project. Below we highlight the most frequently requested strategies.
Reporter Expresses Before Cre Recombination
In the image below we show that a reporter can be expressed together with the gene of interest by adding the reporter to the 3’ end of the gene, and inserting loxP sites to flank both the target exon(s) and the reporter. The reporter will be excised along with the target region so that both the endogenous gene and the reporter are inactivated. This approach is applicable when the gene is small or when the target region is located at the 3’ end of the gene.
Reporter Expresses After Cre Recombination
A reporter gene can also be expressed after Cre recombination has inactivated the gene of interest. This can serve as proof that the gene has been inactivated. Below we show two examples of how this can be achieved:
1. The reporter should not express before Cre recombination due to the endogenous gene’s stop codon and 3’-UTR. The schematic below illustrates that the reporter is placed downstream of the endogenous gene, and is not flanked by loxP sites. The reporter should only express after Cre recombination. This approach is applicable when the gene is small or when the target region is located at the 3’ end of the gene.
2. Here we utilize a more complex approach to accommodate more flexibility on the target region, because the inverted reporter can be placed into an intron downstream of the target exon. In the schematic below we show that the target exon together with the inverted reporter is flanked with loxP sites facing each other. Upon FLP recombination, the Neo cassette is removed, leaving an FRT print behind. Upon Cre recombination, the entire target region between the two loxP sites is inverted, so that the tdTomato reporter is now expressed, whereas exon 2 is inverted and downstream of the reporter’s stop signal. Through this disruption the gene is inactivated. This complex model has recently been published by our client Andrea Meredith at the University of Maryland.
Click on image to enlarge.
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.
For the above described strategies the reporters are expressed from the endogenous gene’s promoter. This is useful to detect when and where the gene expresses, but if the endogenous promoter is not strong enough, the reporter might not be detectable. The consideration of the type of reporter will be important. Some fluorescent reporters have been shown to fluoresce more strongly or be more readily detectable than others, for example BFP and tdTomato. The appropriate reporter gene to use should depend on the detection instrumentation to be used, the nature of the biological sample to be studied (e.g., tissue versus cells), and the type of experimental assay.
We have developed an alternative approach which uses a strong, exogenous promoter to drive the reporter gene, instead of the endogenous promoter of the target gene. Learn more about our No-Doubt-KnockoutTM technology.
For the aforementioned reporter strategies, expression of the reporter, either before or after target gene excision, is driven by the target gene’s endogenous promoter. If the endogenous promoter is not strong enough, the reporter might not be detectable. We have developed an alternative approach for detecting deletion of the target gene which uses a strong, exogenous promoter to drive the reporter gene instead of the endogenous promoter of the target gene. Our technology removes concerns about low expression from the local gene promoter, and provides a reliable visual cue to show that your target gene has been knocked out. Learn more about our No-Doubt-KnockoutTM technology.
Generating one mouse model with the potential for producing multiple derivative mouse lines is a great way to save time and money, as well as control variables across mouse experiments studying the targeted gene of interest. A knockout-first mouse model – with or without a reporter – that can be used in gene rescue experiments, and can also include conditional knockout potential, is a useful mouse model if a researcher wants to study a conventional knockout as well as a conditional knockout of their gene of interest. A reporter can be included for expression when the gene is inactivated globally.
The schematic below illustrates a knockout-first with reporter and conditional knockout potential design. In essence, a Neo-Stop cassette (with or without a reporter gene), including FRT sites and one loxP site, is inserted upstream of the exon(s) to be deleted. A single loxP site is inserted downstream of the target exon(s) to facilitate the conditional knockout potential. Flp-mediated recombination with the FRT sites results in deletion of the Neo-Stop cassette, for rescuing gene expression. The Flp-mediated recombination produces the conditional knockout allele with the target exon(s) flanked by the loxP sites.
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