ROSA26 large fragment knockin strategy design
Tell us the gene you wish to knockin and we will design a nuclease-mediated strategy for you. This includes design of the donor vector and gRNA vectors, and selection of target sites in the gene based on our optimized algorithm that maximizes on-target nuclease activity and minimizes off-target activity. Genotyping assays based on PCR and sequencing will also be designed for the screening of founder mice.
Knockin 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.
CRISPR/Cas9 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 proper translation 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. Cyagen performs up to three rounds of injections to obtain the required number of founders.
Knockin founder screening
Pups will be screened by PCR to identify knockin founder mice. Specifically, the site targeted by CRISPR will be PCR-amplified to reveal any insertions. Occasionally, an animal may be found to have both alleles of the target site mutated.
Breeding founders to obtain F1
Cyagen will breed up to 3 founders to wildtype mice of matching strain background, and genotype their offspring to obtain F1 mice bearing the large-fragment knockin.
ROSA26 large-fragment knockin is provided on a B6 background.
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||ROSA26 large fragment knockin strategy design||Free||1-4 days|
|2||Knockin vector construction (up to 6kb fragment)||$4,400||4-7 weeks|
|3||CRISPR/Cas9 injection to obtain knockin founders||$14,590||8-14 weeks|
|4||Genotyping pups to identify knockin founders||$950||1-2 weeks|
|5||Breeding founders to obtain F1||$2,450||12-16 weeks|
Note: Pricing applied to projects with fragments up to 6kb in length. Larger fragments up to 8kb can be accommodated at an additional cost. Pricing includes up to 3 rounds of injection; additional rounds can be requested for an additional fee. Pricing includes genotyping by PCR; sequencing and southern blot are available upon request for an additional fee. Off-target analysis (up to 5 sites) is available for an additional fee. Please refer to formal quotations for additional information.
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 transgenic 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 or fixed/stained embryos 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.
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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.