Point mutation mice 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 mouse is a knockin mouse line in which one or more nucleotides in mouse 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 models have been widely used to study the roles of particular nucleotides or amino acids in the genetic elements or proteins, and to model human genetic diseases. Cyagen offers quick point mutation mouse model generation via CRISPR/Cas9-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 for you. 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 activity, the design of nuclease vectors and the donor template. Genotyping assays based on PCR and sequencing will also be designed for the screening of knockin founder mice.
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 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 and sequencing to identify knockin founder mice. 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 mice is the end point. However, some customers wish to have us breed the founders further to obtain F1 mice. We will breed up to 3 founders to wildtype mice of matching strain background, and genotype their offspring to obtain F1 mice bearing the knockin allele.
For projects where nuclease expression vectors are constructed by Cyagen
|1||Point mutation strategy design||Free||1-4 days|
|2||Nuclease expression vector construction for point mutation knockin||Please inquire||3-5 weeks|
|3||mRNA preparation||Please inquire||1-2 weeks|
|4||CRISPR/Cas9 injection to obtain point mutation knockin founders||C57BL/6||Please inquire||8-14 weeks|
|5||Genotyping pups to identify point mutation knockin founders||Please inquire||2-4 weeks|
|6||Off-targeting analysis||Please inquire||1-2 weeks|
|7||Breeding founders to obtain F1||Please inquire||12-16 weeks|
Note: For nuclease-mediated point mutation mouse 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 mouse importation, nor transit time during shipping.
We typically produce CRISPR-mediated knockin mice in the C57BL/6 strain background, but we may be able to use other strains per your request.
>> Rat models are also available. Learn more about CRISPR Knockin Rat Services.
Cyagen offers the best guarantee in the industry – we guarantee generation of point mutation knockin mice. 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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