Conditional Knockout Mouse Models
Applied StemCell’s PrimeCKO™ Conditional Knockout (cKO) Mouse Models service will enable you to have precise control over where or when your gene of interest in knocked out. Using CRISPR and the Cre-Lox system, we can generate floxed Conditional Knockout Mouse Models for a variety of temporal and spatial knock-out conditions in mice.
- Affordable! Low prices and founder guarantee
- F1 breeding for germline transmission
- Fast turnaround of 5-8 months
- Novel CRISPR strategy for higher efficiency and success rate
- Animal IP belongs to customers
You can breed the “floxed” PrimeCKO™ conditional knockout mice with commercially available Cre mouse lines or we can generate a custom Cre mouse model for high level Cre expression.
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Choosing the Right Genome Editing Technology for Your Mouse Models: CRISPR, TARGATT™ and Beyond... (February 2017)
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).
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.
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.
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.
We have a greater than 97% success rate in generating a CKO mouse models using CRISPR/Cas9.
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.
The delivery time is around 6-9 months. The cost of the project varies based on the complexity of the modification required. Please inquirefor more details.
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 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.
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.
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CRISPR Knock-in H11 Locus in Pigs
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Knock-in, Knockout, Conditional Knock-out
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- Kweon, S. M., Chen, Y., Moon, E., Kvederaviciutė, K., Klimasauskas, S., & Feldman, D. E. (2019). An Adversarial DNA N6-Methyladenine-Sensor Network Preserves Polycomb Silencing. Molecular Cell. https://doi.org/10.1016/j.molcel.2019.03.018
- Deng, F., He, S., Cui, S., Shi, Y., Tan, Y., Li, Z., ... & Peng, L. (2018). A Molecular Targeted Immunotherapeutic Strategy for Ulcerative Colitis via Dual-Targeting Nanoparticles Delivering miR-146b to Intestinal Macrophages. Journal of Crohn's and Colitis.
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- Besschetnova, T. Y., Ichimura, T., Katebi, N., Croix, B. S., Bonventre, J. V., & Olsen, B. R. (2015). Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis. Matrix Biology, 42, 56-73.
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Homologous Recombination Conditional Knockout Mouse (cited/published articles)
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- Miller, J. N., Kovács, A. D., & Pearce, D. A. (2015). The novel Cln1R151Xmouse model of infantile neuronal ceroid lipofuscinosis (INCL) for testing nonsense suppression therapy. Human Molecular Genetics, 24(1), 185–196. http://doi.org/10.1093/hmg/ddu428.