Standard Conditional Knockout
A standard conditional knockout is a versatile tool for studying your gene of interest – there are as many possible patterns of knockout as there are Cre lines.
Two 34-bp loxP elements are inserted into introns of your gene of interest. When the Cre recombinase enzyme is present it recognizes these elements and causes the deletion of the sequence between them. Ideally the loxP elements flank an exon whose deletion results in a frameshift, leading to early stop codons in the transcript. A conditional knockout mouse model enables the inactivation of a gene of interest in a specific tissue and/or at a specific time point, while in all other tissues the gene retains its wildtype function. Specificity is achieved by combining two elements: target site sequences inserted into the gene of interest and regulated DNA recombinase activity. Most commonly Cre recombinase is used in conjunction with its cognate target site loxP. Using this system, genes with inserted loxP sites will be affected and knockout occurs in cells with recombinase activity. Conditional knockouts have mostly replaced conventional knockouts by avoiding potential embryonic lethality from full-body knockout and giving researchers flexible control over where knockout occurs.
Great flexibility is possible with regards to loxP site placement, depending on the genomic structure of the gene of interest. A region of DNA flanked by loxP sites is said to be “floxed”. For complete knockout two strategies are preferred: creating a frameshift mutation or completely deleting the coding sequence. In many cases the deletion of an early exon creates a frameshift mutation which introduces early stop codons into the sequence. Floxing a region less than 2kb is very straightforward and creates a reliable conditional knockout model. In some cases it is preferable to target a larger region, possibly including most of the coding sequence of the gene. Cre-lox recombination efficiency is reduced as a function of target region size but this is a viable strategy for robust conditional knockout in many genes.
For deletion of the floxed region to occur the mouse carrying the floxed allele is mated with a Cre transgenic mouse 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. 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.
Traditional Conditional Knockout
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. The mRNA produced from this disrupted gene is targeted by the nonsense-mediated decay pathway, generally resulting in no protein being produced. As illustrated below it is also possible for a truncated protein product to be made before early stop codons are encountered.
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)
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 temporally controlled 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.
You will receive a quote within 24 hours, and one of our technical consultants will contact you to discuss your next standard conditional knockout mouse model.