Each full gene targeting project is split into the following phases:
1. Targeting strategy design
2. Targeting vector construction
3. Targeting in ES cells
4. Chimera production
5. F1 heterozygous mutant production (germline transmission)
You can outsource full gene targeting projects to mus, or only the most challenging phases of your projects.
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.
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.
- 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- Humanized mouse: A humanized mouse is defined as a mouse model in which a portion or the full mouse gene sequence has been replaced by its human counterpart.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.
We can work with knockout ES cells from KOMP or EUCOMM. You can obtain ES cells from these organizations yourself and send them to us, or we can obtain the cells on your behalf. We will then produce chimeras or F1 mutants using these cells per your request.
|Targeting strategy design||Free||1-4 days|
|Construction of conventional knockout vector||$5,950||8-14 weeks|
|Construction of conditional knockout or knockin vector||$6,950||8-14 weeks|
|Targeting in ES cells||$8,950||12-20 weeks|
|Optional: Remove drug-selection cassette from conditional knockout or knockin ES cells by FLP or Cre||$1,050/clone||3-5 weeks|
|Optional: Karyotype ES cells to ensure correct chromosome number||$350/clone||1-2 weeks|
(when ES cell clones are generated in-house)
(we will inject as many ES cell clones as needed to fulfill our guarantee)
(when ES cell clones are provided by the customer)
|F1 heterozygous mutant production* (when ES cell clones are generated in-house)||$5,950||10-16 weeks|
|F1 heterozygous mutant production* (when ES cell clones are provided by the customer)||Please inquire|
* 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.
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.
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 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).
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. 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.
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 ES cell mediated Knockout & Knockin Mice
Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis.
The EMBO Journal. 36:2529 (2017)
Nucleic acids are potent triggers for innate immunity. Double‐stranded DNA and RNAadopt different helical conformations, including the unusual Z‐conformation. Z‐DNA/RNA is recognised by Z‐binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM‐1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock‐in models, we report that mutation of key amino acids involved in Z‐DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNAsynthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1‐ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross‐linked to endogenous RNA. These observations show that Z‐RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.