Conditional Knockout Mouse Models

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The development of the CRSIPR/Cas9 nuclease system for genome editing has greatly improved site-directed mutagenesis. This technology has facilitated the generation of accurate and relevant mouse models of human physiology and disease with sophisticated genetic modifications such as conditional/ inducible knockout or expression, humanized mouse models, etc., more efficiently and affordably. In particular, it has enabled the generation of conditional knockout mouse models where the gene of interest is knocked-out only in a tissue of interest, thereby avoiding difficulties posed by constitutive knockout models such as embryonic lethality, undesired phenotypes and compensatory mechanisms.

At Applied StemCell, we can generate custom conditional knockout mouse models specific to your research niche and enable you to have precise control over where or when your gene of interest is knocked-out. You can breed your conditional knockout mouse model with hundreds of commercially available Cre-mice, or we can engineer a custom Cre-mouse line using our complementary and proprietary TARGATT™ technology.

Key Features:

  • Full guarantee for conditional knockout mouse model generation
  • Fast turnaround in as little as 6 months
  • Highly efficient CRISPR protocols and proprietary gRNA validation methods guarantee precise modifications
  • AAALAC-accredited animal facility; all mouse models generated in the USA
  • ISO:9001 certified facility to manufacture CRISPR reagents
  • Affordable rates suitable for academic grants and corporate funding
  • Animal IP belongs to customers
  • Custom Cre- mouse generation available

There are several commercially available Cre mouse models that can be used for crossbreeding with our custom generated CKO models. Most commonly, the Cre expressing mice are generated by traditional homologus recombination which inserts the Cre transgene randomly, thereby contributing to longer deliverables and inefficiency of generating Cre mouse models. At Applied StemCell, we use our TARGATT™ site-specific knock-in technology to knock-in the Cre along with a promoter of choice into a safe harbor locus which guarantees high level Cre expression, but is also a more efficient and affordable technology to generate Cre-mice.

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. 


Technical Details

CRISPR for generating Conditional Knock-out Mice

The most commonly used conditional knockout (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 CKO 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.


CKO 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.

conditional knockout mouse Diagram 2

Diagram 2.  Crossbreeding the CKO 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 causing a frameshift in downstream sequence.


FAQs - Conditional Knockout (CKO) Mouse Models

1. What is the basis of your design algorithm for the sgRNAs?

There are a number of open source tools that have efficient gRNA design capabilities. We cannot share information regarding the design tool we use at Applied StemCell.

2. What is your success rate for generation CRISPR/Cas9 conditional knockout mice?

We have a greater than 97% success rate in generating a CKO mouse models using CRISPR/Cas9.

3. How would you recommend your customers to screen for off-target effects and has that been done for the conditional mice you have produced?

For predicted off-target sites, we can provide a list of the top predicted sites, which you can then evaluate if you choose. If we evaluate off-target sites for a customer, we will typically amplify out several of the top candidates and perform Sanger sequencing. However, especially for CKO models, we generally have a good amount of freedom in the choice of gRNA candidates, and specifically design them to minimize off-target profiles.

4. What is your delivery time and cost for generating a CKO mouse model?

The delivery time is around 6-9 months. The cost of the project varies based on the complexity of the modification required. Please inquire for more details.

5. Do you provide guarantee that the KO will succeed?

We do not offer a guarantee for our CKO projects but our success rate is more than 97%. We can also provide references of researchers who have been satisfied with our CKO mouse model generation service.

Case Studies

Case Studies of CKO Mouse Models Generated Using 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. 

conditional knockout mouse Figure 1

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).

conditional knockout mouse sequencing of PCR fragment

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.   

conditional knockout mouse PCR genotype screening of founder mice

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. 

conditional knockout mouse PCR genotype screening of F1 CKO mice

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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