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.
We typically produce nuclease-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.
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.
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.
If you find another commercial service provider that offers better pricing than ours, we will match the price plus an additional 5% off.
Standard payment terms include a 50% upfront payment before the project, and the remaining 50% plus shipping charge paid after completion of the project. If you need us to design your transgenic strategy, we will provide this service for free irrespective of whether you end up choosing us for your project.
We offer up to a 10% bulk discount for large orders. Large orders are defined as 5 or more projects from the same institution. If you bundle your orders with those of your colleagues, you can all qualify for the bulk discount.
Products are shipped from our Santa Clara, California facility. For mouse shipments, the shipping charge includes courier cost plus a $100/crate handling fee. DNA constructs or fixed/stained embryos are shipped at room temperature, and the charge includes courier cost plus a $10 handling fee. We typically use World Courier to ship live mice and FedEx for other shipments.
All animal work is conducted in our specific pathogen free (SPF) facilities that have been AAALAC accredited and OLAW assured. For details information, please visit our support section for Description of our Facility, Animal Health and Animal Welfare Program.
Please click here to view a map of customers who have used Cyagen before worldwide.
Please click here for a list of publications that have cited Cyagen.
◆ Case studies on our Large-fragment Knockin Mice (ROSA26)
Identification of Natural Regulatory T Cell Epitopes Reveals Convergence on a Dominant Autoantigen.
IMMUNITY. 47:107 (2017)
Leonard JD, Gilmore DC, Dileepan T, Nawrocka WI, Chao JL, Schoenbach MH, Jenkins MK, Adams EJ, Savage PA
Regulatory T (Treg) cells expressing the transcription factor Foxp3 are critical for the prevention of autoimmunity and the suppression of anti-tumor immunity. The major self-antigens recognized by Treg cells remain undefined, representing a substantial barrier to the understanding of immune regulation. Here, we have identified natural Treg cell ligands in mice. We found that two recurrent Treg cell clones, one prevalent in prostate tumors and the other associated with prostatic autoimmune lesions, recognized distinct non-overlapping MHC-class-II-restricted peptides derived from the same prostate-specific protein. Notably, this protein is frequently targeted by autoantibodies in experimental models of prostatic autoimmunity. On the basis of these findings, we propose a model in which Treg cell responses at peripheral sites converge on those self-proteins that are most susceptible to autoimmune attack, and we suggest that this link could be exploited as a generalizable strategy for identifying the Treg cell antigens relevant to human autoimmunity.
The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.
Nat Immunol. 18:214 (2017)
Liu B, Zhang M, Chu H, Zhang H, Wu H, Song G, Wang P, Zhao K, Hou J, Wang X, Zhang L, Gao C
The signaling adaptor MAVS forms prion-like aggregates to activate an innate antiviral immune response after viral infection. However, the molecular mechanisms that regulate MAVS aggregation are poorly understood. Here we identified TRIM31, an E3 ubiquitin ligase of the TRIM family of proteins, as a regulator of MAVS aggregation. TRIM31 was recruited to mitochondria after viral infection and specifically regulated antiviral signaling mediated by RLR pattern-recognition receptors. TRIM31-deficient mice were more susceptible to infection with RNA virus than were wild-type mice. TRIM31 interacted with MAVS and catalyzed the Lys63 (K63)-linked polyubiquitination of Lys10, Lys311 and Lys461 on MAVS. This modification promoted the formation of prion-like aggregates of MAVS after viral infection. Our findings reveal new insights in the molecular regulation of MAVS aggregation and the cellular antiviral response through TRIM31-mediated K63-linked polyubiquitination of MAVS.