Genome Editing Knockout and Knockin Rats

Locate My Expert
Genome Editing Knockout and Knockin Rats
It has not been possible to knockout or knockin genes in the rat until recently. The major game changer was the emergence of several types of artificially constructed nucleases (e.g., Cas9, TALEN, and ZFN), which can be engineered to recognize and cleave arbitrary sequences. When such nucleases (or their DNA or mRNA precursors) designed to target a certain site in the rat genome are microinjected into fertilized rat eggs, cleavage at the site followed by imperfect repair can result in small deletions (and more rarely insertions) of one or more base pairs. If the cut site is in the coding region of a gene, the result can be frameshift mutations downstream of the site. This approach can generate a knockout in as little as ~2 months.
 ◆ Workflow of Genome Editing Knockout & Knockin  Rat Services

Description of Services

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.

Donor Strain Information

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.

Pricing and Turnaround Time

For projects where nuclease expression vectors are constructed by Cyagen

 

Stages Service Price Turnaround time
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.

Guarantee

Cyagen offers the best guarantee in the industry – 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, mobidity, 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, 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 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.

◆ Shipping

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.

◆ 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

Please click here to view a map of customer who have used Cyagen before worldwide. 

◆ Citations

Please click here for a list of publications that have cited Cyagen.