Knockout or knockin strategy design
Tell us the gene you wish to knockout or the point mutation or fragment to knockin, and we will design a nuclease-mediated knockout strategy that meets your research needs. 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 reactivity. Genotyping assays based on PCR and sequencing will also be designed for the screening of founder rats.
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. For each gene, we will construct vectors against at least two target sites in the gene to ensure success.
Nuclease injection into rat eggs
- Preparation of nuclease mRNA: 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 nuclease mRNA will be injected into fertilized rat 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 founders with frameshift mutations or 2 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 300 eggs per target site, and we recommend the targeting of 2-3 sites per gene to increase the probability of success.
Knockout or knockin founder screening
Pups will be screened by PCR and sequencing to identify founder rats. 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. Rats carrying frameshift deletions/insertions on at least one allele are considered knockout founders. Occasionally, an animal can have both alleles of the target site mutated.
Optional: Breeding founders to obtain F1
For most projects, the generation of founder knockout rats is the end point. However, some customers wish to have us breed the founders further to obtain F1 rats. We will breed up to 5 founders to wildtype rats of matching strain background, and genotype their offspring to obtain F1 rats bearing the knockout allele.
We typically produce nuclease-mediated knockout or knockin rats in SD or Long Evans 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 point-mutation knockin strategy design||Free||1-4 days|
|2||Nuclease knockout or knockin expression vector construction (we will construct vectors against at least two target sites in the gene to ensure success)||$1,950||3-5 weeks|
|3||Preparation of nuclease mRNA||$950||1-2 weeks|
|4||Nuclease injection to obtain knockout founders||SD||$15,000||7-12 weeks|
|Long Evans||$17,000||7-16 weeks|
|Nuclease injection to obtain point-mutation knockin founders||SD||$23,950||7-12 weeks|
|Long Evans||$24,950||7-16 weeks|
|5||Founder screening||$950||1-2 weeks|
|6||Off-target analysis||$595||1-2 weeks|
|7||(Optional) Breeding founders to obtain F1||$3,150||12-18 weeks|
Note: For nuclease-mediated knockout rat 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 rat importation and 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, 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 10% bulk discount for large orders. Large orders are defined as 5 or more projects from the same institution. If you bundle your order with that of your colleagues, you can all qualify for the bulk discount.
Products are shipped from our Santa Clara, California facility. For rat 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 rats and FedEx for other shipments.
All animal work is conducted in our specific pathogen free (SPF) facilities that have been AAALAC accredited.
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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.