In order to achieve consistent and predictable ubiquitous/tissue-specific gene expression in your mouse model, targeted insertion into the ROSA26 locus is an ideal choice. The ROSA26 locus is often referred to as a "safe harbor" locus, and has been extensively used as a transgene insertion site due to the lack of insertion site side effects and the stable ubiquitous expression of genes inserted here. Now, with the application of CRISPR/Cas9-based knockin technology, large DNA fragments can be accurately introduced into the ROSA26 locus quickly and efficiently, with turnaround times as short as 3 months. We guarantee delivery of a minimum of 2 founders or 3 F1 animals for ROSA26 knockin with inserted fragment up to 6 kb.
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.
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.
- 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.
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.
For projects where nuclease expression vectors are constructed by Cyagen
|1||Rosa26 large fragment knockin strategy design||Free||1-4 days|
|2||Nuclease expression vector/Knockin vector construction||$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|
|6||(Optional) Southern blotting for Founders or F1||$2,800||2-4 weeks|
Note: Pricing applied to projects with fragments up to 6kb in length. Larger fragments can be accommodated at an additional cost. Please refer to formal quotations for additional information.
We typically produce CRISPR-mediated large fragment knockin (ROSA26 locus) 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 Large-fragment Knockin (ROSA26 Locus) Rat Services.
Cyagen offers the best guarantee in the industry – we guarantee generation of ROSA26 knockin animals with inserted fragment up to 6 kb. Projects with knockin fragment larger than 6 kb will be evaluated on the case by case basis. 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.