• Crispr - Mouse Models

PrimeCKO™ 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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Application Notes

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).

 

SCHEMATIC-CRISPR-CKO-Mouse-v2 
Diagram 1. The schematic describesthe first stage in developing a conditional knock-out mouse model using CRISPR to generate a floxed (loxP flanked exon) mouse. A single stranded donor DNA (ssDNA) is used for delivering the 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. The donor ssDNA is delivered along with Cas9 (mRNA or protein) and validated gRNAs via microinjection.

 

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
What is the basis of your design algorithm for the sgRNAs?
What is your success rate for generation CRISPR/Cas9 conditional knockout mice?
How would you recommend your customers to screen for off-target effects and has that been done for the conditional mice you have produced?
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Do you provide guarantee that the KO will succeed?
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. 

Publications

CRISPR Knock-in, CRISPR Knockout Mouse

CRISPR Technology

CRISPR Knock-in H11 Locus in Pigs

  • Ruan, J., Li, H., Xu, K., Wu, T., Wei, J., Zhou, R., ... & Chen-Tsai, R. Y. (2015). Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Scientific reports, 5, 14253.

 Knock-in, Knockout, Conditional Knock-out

 

  • 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.
  • Jo, S., Fonseca, T. L., Bocco, B. M. D. C., Fernandes, G. W., McAninch, E. A., Bolin, A. P., ... & Németh, D. (2018). Type 2 deiodinase polymorphism causes ER stress and hypothyroidism in the brain. The Journal of Clinical Investigation.
  • Langston, R. G., Rudenko, I. N., Kumaran, R., Hauser, D. N., Kaganovich, A., Ponce, L. B., ... & Beilina, A. (2018). Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2. Neurochemical research, 1-14.
  • Amara, N., Tholen, M., & Bogyo, M. (2018). Chemical tools for selective activity profiling of endogenously expressed MMP-14 in multicellular models. ACS Chemical Biology. doi: 10.1021/acschembio.8b00562.
  • Allocca, S., Ciano, M., Ciardulli, M. C., D’Ambrosio, C., Scaloni, A., Sarnataro, D., ... & Bonatti, S. (2018). An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population. International journal of molecular sciences, 19(7).
  • Peng, L., Zhang, H., Hao, Y., Xu, F., Yang, J., Zhang, R., ... & Chen, C. (2016). Reprogramming macrophage orientation by microRNA 146b targeting transcription factor IRF5. EBioMedicine, 14, 83-96.
  • Hu, J. K., Crampton, J. C., Locci, M., & Crotty, S. (2016). CRISPR-mediated Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ triple gene disruption reveals NKT cell defects but not T follicular helper cell defects. PloS one, 11(5), e0156074.
  • 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.
  • McKenzie, C. W., Craige, B., Kroeger, T. V., Finn, R., Wyatt, T. A., Sisson, J. H., ... & Lee, L. (2015). CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice. Molecular biology of the cell, 26(18), 3140-3149.
  • Bishop, K. A., Harrington, A., Kouranova, E., Weinstein, E. J., Rosen, C. J., Cui, X., & Liaw, L. (2016). CRISPR/Cas9-mediated insertion of loxP sites in the mouse Dock7 gene provides an effective alternative to use of targeted embryonic stem cells. G3: Genes, Genomes, Genetics, 6(7), 2051-2061.

Homologous Recombination Conditional Knockout Mouse (*cited/published articles)

  • Li, C., Zheng, Z., Ha, P., Chen, X., Jiang, W., Sun, S., ... & Chen, E. C. (2018). Neurexin Superfamily Cell Membrane Receptor Contactin‐Associated Protein Like‐4 (Cntnap4) is Involved in Neural EGFL Like 1 (Nell‐1)‐responsive Osteogenesis. Journal of Bone and Mineral Research https://doi.org/10.1002/jbmr.3524.
  • Geraets, R. D., Langin, L. M., Cain, J. T., Parker, C. M., Beraldi, R., Kovacs, A. D., ... & Pearce, D. A. (2017). A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies. PloS one, 12(5), e0176526
  • Miller, J. N., Kovács, A. D., & Pearce, D. A. (2015). The novel Cln1R151Xmouse model of infantile neuronal ceroid lipofuscinosis (INCL) for testing nonsense suppression therapyHuman Molecular Genetics24(1), 185–196. http://doi.org/10.1093/hmg/ddu428.
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