Point mutation rats are used to investigate the roles of specific nucleotides in gene function and are often used to study genetic disorders and rare diseases. A point mutation rat is a knockin rat line in which one or more nucleotides in rat genome are substituted by variant nucleotides. This can result in either an in-frame amino acid change of protein sequence or a frameshift mutation. Point mutation knockin rat models are of particular interest in that they mimic human diseases better than mouse models. Cyagen offers quick point mutation rat model generation via CRISPR-Pro-mediated genome editing technology by co-injecting nuclease and oligo donors carrying the desired mutation and homology arms. We guarantee delivery of a minimum of 2 founders or 3 F1 animals for point mutation knockin.
Tell us the name of your gene and the point mutation desired to knockin, 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, the design of nuclease vectors and the donor template. Genotyping assays based on PCR and sequencing will also be designed for the screening of founder rats.
DNA vectors that express the desired nucleases will be constructed. Where needed, the efficacy of these vectors will be tested in cell culture.
- mRNA preparation: 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 in vitro transcribed nuclease mRNA and oligo donor carrying the desired mutation(s) will be co-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.
For some projects, the generation of founder 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 knockin allele.
For projects where nuclease expression vectors are constructed by Cyagen
|Point mutation strategy design
|Nuclease expression vector construction for point mutation knockin
|CRISPR-Pro injection to obtain point mutation knockin founders
|Genotyping pups to identify point mutation knockin founders
|Breeding founders to obtain F1
Note: For nuclease-mediated point mutation 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, nor transit time during shipping.
We typically produce CRISPR-mediated knockin rats in SD or Long Evans strain background, but we may be able to use other strains per your request.
>> Mouse models are also available. Learn more about CRISPR Knockin Mouse Services.
Cyagen offers the best guarantee in the industry – we guarantee generation of point mutation knockin rats. 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.
Please see below for supporting information related to Cyagen animal orders.
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