Genome editing using or CRISPR/Cas9 or TALEN can generate a knockout or knockin in as little as ~2 months, far faster than the typical 8-12 months required for conventional knockouts, because there is no need for ES cell work or chimera production.
Knockout or knockin strategy design
Tell us the gene you wish to knockout or the point mutation desired to knockin and we will design a nuclease-mediated strategy for you. This includes identification of the type of nuclease (CRISPR/Cas9 or TALEN) most suitable for your goals, and the selection of target sites in the gene based on our optimized algorithm that maximizes on-target nuclease activity and minimizes off-target activity. 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 or knockin founder mice.
Nuclease 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.
- Pronuclear injection to obtain founders: The nuclease mRNA 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 constructed by Cyagen, we guarantee a minimum of 2 knockout or knockin founders. We will target as many sites and inject as many eggs as needed to fulfill this guarantee. In cases where the nuclease expression vectors (or their mRNA products) are provided by the customer, we will inject a minimum of 200 eggs (if FVB strain is used) or 300 eggs (if B6 strain is used) per target site. We recommend the targeting of 2-3 sites per gene to increase the probability of success.
Knockout/Knockin founder screening
Pups will be screened by PCR and sequencing to identify knockout or knockin 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 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 most 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 5 founders to wildtype mice of matching strain background, and genotype their offspring to obtain F1 mice bearing the knockout or knockin allele.
We typically produce nuclease-mediated knockout or knockin mice in the B6 strain background, but we may be able to use other strains per your request.
Cyagen will fully refund the client’s service fee if animals specified in the project are not generated – the best guarantee in the industry (except for genetic modifications severely affecting viability, morbidity, or fertility). If Cyagen is unable to provide specified animals and the client wishes to obtain any upstream products (e. g. nuclease vector), the client must pay per milestone for services performed to generate said upstream products, plus shipping and handling.
For projects where nuclease expression vectors are constructed by Cyagen
|1||Knockout or knockin strategy design||Free||1-4 days|
|2||Nuclease expression vector construction for knockout||$1,950||3-5 weeks|
|Nuclease expression vector construction for point mutation knockin||$2,450||3-5 weeks|
|3||mRNA preparation||$950||1-2 weeks|
|4||CRISPR/Cas9 or TALEN injection to obtain knockout founders||FVB||$5,950||6-8 weeks|
|CRISPR/Cas9 or TALEN injection to obtain point mutation knockin founders||FVB||$9,450||8-14 weeks|
|5||Genotyping pups to identify knockout founders||$950||1-2 weeks|
|Genotyping pups to identify point mutation knockin founders||$1,450||2-4 weeks|
|6||Off-target analysis||$595||1-2 weeks|
|7||Breeding founders to obtain F1||$2,450||12-16 weeks|
For projects where nuclease expression vectors are provided by the customer
|1||Nuclease expression vector material handling fee||Please Inquire||N/A|
|2||mRNA preparation||$950/target site||1-2 weeks|
|3||Nuclease injection to obtain founders||FVB||$3,950/target site||6-8 weeks|
|4||Nuclease injection to obtain founders||C57BL/6||$6,450/target site||6-10 weeks|
|5||Knockout founder screening||$950/target site||1-2 weeks|
|6||Off-target analysis||$595/target site||1-2 weeks|
|7||Breeding founders to obtain F1||$1,250/target site||12-16 weeks|
Note: For nuclease 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.
If you find another commercial service provider that offers better pricing than ours, we will match the price plus an additional 5% off.
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.
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.
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.
All animal work is conducted in our specific pathogen free (SPF) facilities that have been AAALAC accredited.
Our animal health status is on par with or exceeds the requirements of most animal facilities at major research institutions. Animals are tested quarterly for a range of pathogens. Please click here for a list of pathogens excluded from our facilities.
Please click here to view a map of customer who have used Cyagen before worldwide.
Please click here for a list of publications that have cited Cyagen.
We consider all reagents and animals resulting from our services to be the customers’ property. All information regarding our services, including customer identity and contact information, is kept confidential.