ROSA26 large fragment knockin strategy design
Tell us the name of 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 and donor vector 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.
- Nuclease injection to obtain founders: The nuclease mRNA and donor vector will be co-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 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 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
For some 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. 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.
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.