Genome Editing Knockout and Knockin Mice

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Genome Editing Knockout and Knockin Mice
If you wish to quickly knockout or knockin a gene in the mouse, you should consider nuclease-mediated genome editing technologies as an alternative to the conventional, recombination-based knockout approach. Several types of artificially constructed nucleases (e.g. Cas9, TALEN and ZFN) can be engineered to recognize and cleave arbitrary sequences. When such nucleases (or their DNA or mRNA precursors) designed to target a specific site in the mouse genome are microinjected into fertilized mouse eggs, cleavage at the target site followed by imperfect repair can result in small deletions (and insertions, more rarely) of one or more base pairs. If the cut site is in the coding region of a gene, this will result in frameshift mutations downstream of the site, generating a knockout. If a repair template with homology to the target site is present during the repair process, specific point mutations in the repair template can be introduced at the nuclease cleavage site, generating a knockin.

Genome editing using or CRISPR/Cas9 or TALEN can generate a knockout or knockin in as little as ~2 months, far faster than the typical 8-12 months required for conventional knockouts, because there is no need for ES cell work or chimera production.

 Workflow of Gene Editing Mouse Services

Description of Services

Knockout or knockin strategy design

Tell us the gene you wish to knockout or the point mutation desired to knockin and we will design a nuclease-mediated strategy for you. This includes identification of the type of nuclease (CRISPR/Cas9 or TALEN) most suitable for your goals, and the selection of target sites in the gene based on our optimized algorithm that maximizes on-target nuclease activity and minimizes off-target activity. For each gene to be knocked out, we will design vectors against at least two target sites in the gene to ensure success. Genotyping assays based on PCR and sequencing will also be designed for the screening of knockout or knockin founder mice.

 

Nuclease expression vector construction

DNA vectors that express the desired nucleases will be constructed. Where needed, the efficacy of these vectors will be tested in cell culture.

 

Nuclease 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 its proper translation into protein in mammalian cells.

 

- Pronuclear injection to obtain founders: The nuclease mRNA will be 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 constructed by Cyagen, we guarantee a minimum of 2 knockout or knockin founders. We will target as many sites and inject as many eggs as needed to fulfill this guarantee. In cases where the nuclease expression vectors (or their mRNA products) are provided by the customer, we will inject a minimum of 200 eggs (if FVB strain is used) or 300 eggs (if B6 strain is used) per target site. We recommend the targeting of 2-3 sites per gene to increase the probability of success.

 

Knockout/Knockin founder screening

Pups will be screened by PCR and sequencing to identify knockout or 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. Mice carrying frameshift deletions/insertions on at least one allele are considered knockout founders. Occasionally, an animal may be found to have both alleles of the target site mutated.

 

Breeding founders to obtain F1

For most 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 5 founders to wildtype mice of matching strain background, and genotype their offspring to obtain F1 mice bearing the knockout or knockin allele.

Donor Strain Information

We typically produce nuclease-mediated knockout or knockin mice in the B6 strain background, but we may be able to use other strains per your request.

Pricing and Turnaround Time

For projects where nuclease expression vectors are constructed by Cyagen

Stages Service Price Turnaround time
1 Knockout or knockin strategy design Free 1-4 days
2 Nuclease expression vector construction for knockout $1,950 3-5 weeks
Nuclease expression vector construction for point mutation knockin $2,450 3-5 weeks
3 mRNA preparation $950 1-2 weeks
4 CRISPR/Cas9 or TALEN injection to obtain knockout founders FVB $5,950 6-8 weeks
C57BL/6 $9,950 6-10 weeks
CRISPR/Cas9 or TALEN injection to obtain point mutation knockin founders FVB $9,450 8-14 weeks
C57BL/6 $13,450 8-14 weeks
5 Genotyping pups to identify knockout founders $950 1-2 weeks
Genotyping pups to identify point mutation knockin founders $1,450 2-4 weeks
6 Off-target analysis $595 1-2 weeks
7 Breeding founders to obtain F1 $2,450 12-16 weeks

 

For projects where nuclease expression vectors are provided by the customer

Stages Service Price Turnaround time
1 Nuclease expression vector material handling fee Please Inquire N/A
2 mRNA preparation $950/target site 1-2 weeks
3 Nuclease injection to obtain founders FVB $3,950/target site 6-8 weeks
4 Nuclease injection to obtain founders C57BL/6 $6,450/target site 6-10 weeks
5 Knockout founder screening $950/target site 1-2 weeks
6 Off-target analysis $595/target site 1-2 weeks
7 Breeding founders to obtain F1 $1,250/target site 12-16 weeks

Note: For nuclease 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.

Guarantee

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, mobidity, 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.

Inquiries and Quote Requests

Request a quote now. Alternatively, you can always email service@cyagen.com or call 800-921-8930 to inquire about our services or obtain a quote for your project.

◆ Price Matching

If you find another commercial service provider that offers better pricing than ours, we will match the price plus an additional 5% off.

◆ Payments

Standard payment terms include a 50% upfront payment before the project begins, and the remaining 50% plus shipping charge paid after completion of the project. If you need us to design your knockout strategy, we will provide this service for free irrespective of whether you end up choosing us for your project. 

Bulk Discount

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.

◆ Shipping

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 in E. coli 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.

◆ Animal Programs

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.

◆ Customer References

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◆ Citations

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