Knockout strategy design
Tell us the name of gene you wish to knockout, and we will design a nuclease-mediated 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, and the design of nuclease expressing vector(s). For each gene, 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 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.
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(s) 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 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.
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 or critical exon(s) deletion on at least one allele are considered knockout founders. Occasionally, an animal can have both alleles of the target site mutated.
Breeding founders to obtain F1
For some 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 3 founders to wildtype rats of matching strain background, and genotype their offspring to obtain F1 rats bearing the knockout allele.
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, 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.