ES cell mediated Knockout & Knockin Mice

We offer a one-stop solution for all your gene targeting mouse model needs. Our services include conditional knockout, conventional knockout, and knockin. You can contract us to perform your entire targeting project from initial strategy design and targeting vector construction to mutant animal delivery, or you can outsource specific phases of the project to us. Our turnaround time is the shortest in the industry, our prices are unbeatable, and for full gene targeting projects, we guarantee the generation of animals, or your money back.

 

 ◆ Workflow of Gene Targeting Mouse Services

Each full gene targeting project is split into the following phases:

1. Targeting strategy design

• Analyze relevant information for your target gene, including gene structure, neighboring genes at the genomic locus, known targeted animal models, etc.
• Develop a gene targeting strategy, including the method for screening targeted ES cells by PCR and confirmation by Southern blot.
• Propose the strategy to you for review and approval. 

 

2. Targeting vector construction

• Clone relevant DNA fragments corresponding to homology arms into the targeting vector.
• Confirm construction of the final targeting vector by restriction digest and sequencing.

 

3. Targeting in ES cells

• Electroporate linearized targeting vector into ES cells, followed by appropriate drug selection, and isolation of drug-resistant clones. We can use ES cells derived from the C57BL/6 (B6) or BALB/c strains by default, but you can inquire about using ES cells from other strains such as 129.
• Screen for targeted ES cell clones by PCR.
• Expand up to six PCR-positive clones, which are further confirmed by Southern blot.
• Optional: Remove drug-selection cassette in conditional knockout or knockin ES cells by FLP or Cre.
• Optional: Karyotype ES cells to ensure correct chromosome number.

 

4. Chimera production

• Use either injection or aggregation to introduce targeted ES cells into host embryos followed by transfer into surrogate mothers.
• Score resulting pups for coat color chimerism.

 

 

5. F1 heterozygous mutant production (germline transmission)

• Breed male chimeras to wild-type females.
• Genotype pups by PCR to identify F1 heterozygous mutants.

 

You can outsource full gene targeting projects to us, or only the most challenging phases of your projects.

◆ Types of Gene Targeting Mouse Services 

Conventional knockout
In conventional knockout models, one or more critical exons of the target gene are replaced with a drug-selection cassette. This results in the permanent inactivation of the target gene in all cells of the body throughout development.

 

 

 

Conditional knockout
In conditional knockout models, one or more critical exons of the target gene are flanked by LoxP sequences which can be recognized by Cre recombinase. By breeding these floxed mice (mice with gene locus flanked by LoxP) with tissue-specific Cre-expressing mice (Cre deleters), it is possible to specifically delete the floxed region and inactivate the gene in desired tissues, while in all other tissues, the target gene remains functional. 

 

 

 

Knockin 
- Reporter knockin: A visually detectable marker gene (such as GFP or lacZ) is knocked into a gene locus to replace the coding sequence of the target gene for the purpose of monitoring the promoter activity of the gene. Alternatively, a marker or an epitope tag can be knocked into the end (or less frequently, the beginning) of the coding sequence to form a fusion protein, which allows target gene expression and localization/trafficking to be examined.

 

 

 

- Point mutation knockin: By introducing point mutation(s) into homology arms or the targeting vector, desired mutation(s) can be incorporated into the target gene. The mutated gene is expressed under the control of the wildtype gene regulatory elements. 

 

 

 

- Humanized mouse: A humanized mouse is defined as a mouse model in which a portion or the full mouse gene sequence has been replaced by its human counterpart. The human gene is expressed under the control of the wildtype mouse regulatory sequences.

 

 

 

- ROSA26 knockin: ROSA26 is a locus that has been widely used for achieving global expression of the introduced gene in mice. The gene of interest is targeted into the first intron of this locus. The expression of the gene can be driven by the endogenous ROSA26 promoter or by a promoter of choice that is targeted into the locus along with the gene. 

 

 

◆ Pricing and Turnaround Time
 

 

* For a conditional knockout or knockin, the drug-selection cassette can be removed in F1 by crossing chimeras to FLP or Cre deleter mice.

Note: For knockout/knockin 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 and 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, 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.

◆ Inquiries and Quote Requests

Request a quote now. Alternatively, you can always email animal-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 paid after completion of the project. If you need us to design your targeting strategy, we will provide this service for free irrespective of whether you end up choosing us for your project (other companies typically charge a few thousand dollars for this service).

◆ 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 facility in China to our Santa Clara, California facility, then are relayed to end users. For mouse shipments, the shipping charge includes courier cost plus a $100/crate handling fee. Cells are shipped on dry ice, and the charge includes courier cost plus a $45 handling fee. DNA constructs are shipped in E. coli 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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