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Knockout Mice

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rapid knockout with crispr technology
In the past two decades, hundreds of knockout mouse lines have been created by homologous recombination in ES cells, and these mouse lines have been playing pivotal roles not only in determining gene functions in one or more cellular processes, but also in gaining important insights into mechanisms that underline human diseases, as well as in evaluating compounds to speed up drug discovery process.

Engineered nuclease-mediated genome editing (especically CRISPR/Cas9) is an emerging technology which can serve as an alternative to the conventional, ES cell homologous recombination-based knockout animal generation. When gRNA(s) designed to target specific site(s) in the mouse genome and Cas9 are co-injected into fertilized mouse eggs, cleavage at the target site(s) followed by imperfect repair can result in indels (insertion or deletion). If the cut site is in the coding region of a gene, this may result in a frameshift mutation downstream of the site, generating a constitutive knockout. If deletion of exon(s) encoding critical domains is desirable, two gRNAs targeting sites upstream and downstream of the exon(s) can be co-injected with Cas9 and knockout pups with critical region deletion can be generated. Genome editing using CRISPR/Cas9 system can generate constitutive knockout founder animals in as little as 3 months, far faster than the typical 8~12 months required for conventional knockout mice generation with ES cell homologous recombination. We guarantee delivery of a minimum of 2 founders or 3 F1 animals for knockout.

 

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 Workflow of CRISPR/Cas9-mediated Knockout Mouse Services

workflow of CRISPR/Cas9-mediated knockout mouse services

Description of Services

Knockout strategy design

Tell us the name of gene you wish to knockout and we will design a nuclease-mediated strategy for you. This includes the selection of target sites in the gene based on our optimized algorithm that maximizes on-target nuclease activity and minimizes off-target activity, and the design of nuclease expression vector(s). For each gene to be knocked out, we will design vectors against at least two target sites in the gene to ensure success. Genotyping assays based on PCR and sequencing will also be designed for the screening of knockout founder mice.

 

Nucleases expression vector construction

DNA vectors that express the desired nucleases will be constructed. Where needed, the efficacy of these vectors will be tested in cell culture.

 

Nuclease injection into mouse eggs

- mRNA preparation: Nuclease expression vectors will be transcribed in vitro. The resulting mRNA will be artificially capped and polyadenylated to facilitate its proper translation into protein in mammalian cells.

 

- Nuclease injection to obtain founders: The in vitro transcribed nuclease mRNA(s) will be injected into fertilized mouse eggs, followed by implantation of the eggs into surrogate mothers to obtain offspring. In cases where the nuclease expression vectors are designed and constructed by Cyagen, we will inject as many eggs and/or target as many sites as needed to fulfill the guarantee. In cases where the nuclease expression vectors (or their mRNA products) are provided by the customer, we will inject a minimum of 200/300 eggs (based on strain) and screen pups for founders carrying desired mutation. If no founders are identified, more injections can be performed at an additional charge.

 

Founder screening

Pups will be screened by PCR and sequencing to identify knockout founder mice. Specifically, the site targeted by the nucleases will be PCR-amplified, followed by sequencing of the PCR product to reveal any mutations that might have occurred. Mice carrying frameshift deletions/insertions or critical exon(s) deletion on at least one allele are considered knockout founders. Occasionally, an animal may be found to have both alleles of the target site mutated.

 

Breeding founders to obtain F1

For some projects, the generation of founder mice is the end point. However, some customers wish to have us breed the founders further to obtain F1 mice. We will breed up to 3 founders to wildtype mice of matching strain background, and genotype their offspring to obtain F1 mice bearing the knockout allele.

Donor Strain Information

We typically produce CRISPR-mediated knockout mice in the C57BL/6 and FVB background, but we may be able to use other strains per your request.

Pricing and Turnaround Time

For projects where nuclease expression vectors are constructed by Cyagen

Stages Service Price Turnaround time
1 Knockout strategy design Free 1-4 days
2

Nuclease expression vector construction for knockout

$1,950 3-5 weeks
3 mRNA preparation $950 1-2 weeks
4 CRISPR/Cas9 injection to obtain knockout founders FVB $5,950 6-8 weeks
C57BL/6 $9,950 6-10 weeks
5 Genotyping pups to identify knockout founders $950 1-2 weeks
6 Off-target analysis $595 1-2 weeks
7 Breeding founders to obtain F1 $2,450 12-16 weeks

 Note: For nuclease-mediated knockout mouse services not listed above, please inquire about availability and pricing. The turnaround time above does not include the time for obtaining host institution’s approval for mouse importation, nor transit time during shipping.

Guarantee

Cyagen offers the best guarantee in the industry – we guarantee generation of constitutive knockout mice. We will fully refund the client’s service fee if animals with the specified genotype are not generated (except for genetic modifications severely affecting viability, morbidity, or fertility). Given the complexity of biological systems, a particular genetic modification may not result in the desired phenotype. As such, Cyagen's guarantee covers the creation of animals with the specified genotype, not a particular phenotypic outcome in terms of transcription, protein/RNA function, or organismal biology.

Inquiries and Quote Requests

Request a quote now. Alternatively, you can always email service@cyagen.com or call 800-921-8930 to inquire about our services or obtain a quote for your project.

◆ Price Matching

If you find another commercial service provider that offers better pricing than ours, we will match the price plus an additional 5% off.

◆ Payments

Standard payment terms include a 50% upfront payment before the project begins, and the remaining 50% plus shipping charge paid after completion of the project. If you need us to design your knockout strategy, we will provide this service for free irrespective of whether you end up choosing us for your project. 

Bulk Discount

We offer up to a 10% bulk discount for large orders. Large orders are defined as 5 or more projects from the same institution. If you bundle your orders with those of your colleagues, you can all qualify for the bulk discount.

◆ Shipping

Products are shipped from our Santa Clara, California facility. For mouse shipments, the shipping charge includes courier cost plus a $100/crate handling fee. DNA constructs in E. coli are shipped at room temperature, and the charge includes courier cost plus a $10 handling fee. We typically use World Courier to ship live mice and FedEx for other shipments.

◆ Animal Programs

All animal work is conducted in our specific pathogen free (SPF) facilities that have been AAALAC accredited and OLAW assured. For details information, please visit our support section for Description of our Facility, Animal Health and Animal Welfare Program.

◆ Customer References

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◆ Citations

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