Conditional Knock-Out Mouse Models

Applied StemCell’s comprehensive service platform for generating conditional knockout mice provides:

  • Site-specific, floxed alleles (two flanking loxP sites) insertion using CRISPR
  • Highly efficient Cre knock-in, under control of any promoter of choice, using our proprietary TARGATT™ technology
  • The ability to avoid drawbacks of constitutive knock-out models such as embryonic lethality, compensatory mechanisms and undesired phenotypes
  • Generation of conditional knockout mice in as little as 6 months.

CRISPR for generating Conditional Knock-out Mice

The CRISPR-Cas9 system is a revolutionary technology that has advanced site specific genome editing while at the same time simplifying the technology for ease of use and a fast turnaround. It is a versatile editing platform for generating knock-in, knock-out and point mutations models in animals and cells and also for generating a conditional knockout model. The advantage of CRISPR/Cas9 over ZFNs or TALENs is its scalability and multiplexibility in that multiple sites within the mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing in mammalian cells.

Conditional knockout (CKO) animal models are gaining popularity as they circumvent the impediments of constitutive knockout models such as embryonic lethality, compensatory mechanisms and undesired phenotypes and model human diseases better. The most commonly used CKO system is the Cre-LoxP system, where the gene of interest (targeted exons) is flanked by two LoxP sequences (also called floxed allele). The flanking LoxP sequences are inserted at specific sites on either side of the gene of interest using CRISPR technology. The LoxP sites are a target for the Cre Recombinase which catalyzes the deletion of the floxed exon(s).

Diagram 1. The schematic describes the strategies in developing a conditional knock-out mouse model using CRISPR-Cas9 technology. In Strategy1, a donor plasmid is used to deliver floxed targeting exons to replace the wildtype form. The donor contains two LoxP sequences flanking the targeted exon(s) along with 5' and 3' homologous arms for directing a site specific homology directed repair. In Strategy 2, two separate CRISPR systems are designed to insert the two LoxP sequences at the desired 5' and 3' locations to flank the targeting exons.


Conditional knockout mice are generated by crossbreeding two transgenic mouse lines, one with homozygous “floxed” (flanked by loxP) allele, and the other bearing Cre recombinase transgene under the control of a promoter directing tissue specific expression or ubiquitous expression. The Cre expression has minimal unwanted effects in the animal as the mouse genome does not contain endogenous loxP sites, providing an ideal background for site-specific recombination.


Diagram 2.  Crossbreeding the conditional knock-out mouse with a Cre-recombinase expressing mouse. The Cre expression is driven by a promoter of choice: a tissue specific or ubiquitous promoter. The expressed Cre recombinase deletes the floxed exon(s) in a tissue specific manner there by casuing a frame shift in downstream sequence.

Most commonly, the Cre expressing mice are generated by conventional homologus recombination which inserts the Cre transgene randomly, thereby contributing to longer deliverables and inefficiency of generating Cre mouse models.

There are several Cre mouse models available at Jackson labs that can be used for crossbreeding with our custom generated conditional knock-out models.

Can't find a Cre mouse model that fits your research needs? Contact us to see how we can generate a LoxP and Cre breeding pair for you. Applied StemCell's proprietary TARGATT™ knock-in mouse technology enables highly efficient Cre knock-in, under control of any promoter of choice.


Case Studies

Case Studies of Conditional Knock-out Mouse Models generated by Applied StemCell's CRISPR Technology

Case Study #1: A conditional knock-out mouse model with LoxP sequences inserted in intron 1 and downstream of 3’ UTR of the desired locus.

This conditional knockout mouse model was generated using CRISPR Technology by inserting LoxP sequences in intron 1 and downstream of 3’ UTR of the gene of interest . In the first step, a  mixture of active guide RNA molecules (gRNAs), two single stranded oligo donor nucleotide (ssODN) and qualified Cas-9 mRNA was prepared and injected into cytoplasm of C57BL/6 embryos. The second step was to screen new mice born from the microinjection for the presence of LoxP sites at designated locations using PCR. And the third step was to confirm the potentially positive animals by sequencing the modified regions in the mouse genomic locus. 


Figure 1. PCR results of mice born after microinjection of the embryos with CRISPR cocktail. Two out of twelve mice were identified as founders and showed the expected fragment shifts for both 5’ and 3’ LoxP insertions. A LoxP insertion at the 5’site, or intron 1 produced a 513bp PCR fragment (blue box; WT: 473bp) and LoxP insertion at the 3’-targeting site produced a 539bp PCR fragment (red box; WT: 499 bp).


Figure 2. Representative illustration sequence analyses of founder mice confirms LoxP insertion at 5’ and 3’ location at the desired genome locus.

Case Study# 2: Generation of a conditional mouse models with a floxed exon using CRISPR

This conditional knockout mouse (CKO) model was generated using CRISPR technology by inserting two LoxP cassettes on either side of the exon to be conditionally removed. The exon can then be removed by crossbreeding the floxed mouse with a Cre-recombinase-expressing mouse. We generated the floxed model using three well optimized steps: (1) a mixture of two sets of active guide RNA molecules (gRNAs), two single stranded oligodeoxylnucleotides (ssODNs) and qualified Cas-9 mRNA was injected into the cytoplasm of C57BL/6 embryo; (2) new mice born from microinjection were screened using a scheme combining PCR and restriction enzyme digest; (3) we confirmed the floxed allele positive animals by sequencing the modified region in the mouse locus.   

Figure 1. PCR genotype screening of founder mice. (a) PCR product size shift for 5’LoxP and 3’LoxP insertions; (b) Chromatograms of 5’LoxP and 3’LoxP PCR fragments from founder mice. Genomic DNA extracted from individual mice born from microinjection of the embryos were subjected to genotyping PCR. Two mice were identified as potential founders. Sequencing results showed that both mice have LoxP insertions and that mouse# 1 may have 5’ LoxP on both alleles. 

Figure 2. PCR genotype screening of F1 CKO mice. (a) PCR product size shows fragment size shift for 5’LoxP and 3’LoxP insertions in 9 mice (#1, 5, 8, 11, 13, 15; 16, 17 and 20 (b) Chromatograms of 5’LoxP and 3’LoxP sequences at correct location in genome. 


Miller, JN., et al. (2015). Human molecular genetics, 24(1), 185-196.

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