In conditional knockout mice, a gene is inactivated in a specific tissue to study the function of individual genes and model human diseases.
Our TurboKnockout® service can provide you with knockout and conditional knockout (flox) mouse models in as fast as 6-8 months. For conditional knockouts, it may be possible for Cyagen to breed founders directly to your tissue-specific Cre mice or even develop the Cre strains necessary for your study.
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.
Conventional/Constitutive Knockout (KO) Strategy Example
Although conditional knockout (cKO) mouse models are used in similar applications to KO models, conditional models provide precise control of gene expression to yield greater experimental capabilities. Compared with constitutively expressed gene mutations, conditional models provide greater temporal and regional control of gene expression.
The Cre-Lox recombination system enables the generation of tissue-specific or inducible knockouts with a high level of control over the spatial and temporal expression of genes. In the Cre-Lox model development strategy, one or more critical exons of the target gene are flanked by LoxP sequences – known as the floxed gene - which can be recognized by Cre recombinase. By breeding floxed mice with tissue-specific Cre-expressing mice (Cre deleter strain), it is possible to specifically delete the floxed region and inactivate the gene in desired tissues, while the target gene remains functional in all other tissues.
Gene Floxing (Cre-LoxP) Strategy Example
The ‘knockout-first’ (KO-first) allele overcomes the limitations of constitutively-expressed mutations, flexibly producing reporter knockouts, conditional knockouts, and null alleles through exposure to site-specific cyclization recombination (Cre) and flippase (FLP) recombinases – such as in crosses to FLP and cre mice. Knockout-first alleles can also be readily modified in cells using dual recombination-mediated cassette exchange. This strategy relies on the identification of a 'critical' exon common to all transcript variants that, when deleted, creates a frame-shift mutation.
Knockout-First (KO-First) Strategy Example
The KO-first strategy has implications for functional genomics and proteomics across many model systems, including mouse, rat, and human pluripotent stem cells – serving as a functional genomics platform enabling systematic, genome-scale programs for proteomic mapping. The KO-first allele design also forms the basis for generation of lacZ-tagged conditional alleles as part of the International Knockout Mouse Consortium (IKMC) effort organized by the International Mouse Phenotyping Consortium (IMPC).
|Service||Price (Standard B6 strains)||Turnaround time|
|TurboKnockout® targeting strategy design||Free||1-4 days|
|Construction of TurboKnockout® gene targeting vector||Please inquire||8-14 weeks|
|Gene targeting in ES cells||Please inquire||8-14 weeks|
|Karyotype ES cells to ensure correct chromosome number||Please inquire||1-2 weeks|
|TurboKnockout® mouse production||Please inquire
(we will inject as many ES cell clones as needed to fulfill our guarantee)
|Optional: Breeding founders to obtain F1||Please inquire*|
* You can choose to receive F0 founder animals and perform their own breeding to F1 mice. To ensure the successful generation of F1 mice and germline transmission, breeding of F0 animals require adherence to Turboknockout® breeding protocol, which will be supplied to you. You can also choose to have Cyagen perform F1 breeding, which adds 1-3 months to the projected turnaround time. You can choose to breed F0 founders to wildtype mice or to other stains such as Cre deleter strains of your choice; additional service fees may apply.
Note: For special 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.
Mouse: C57BL/6, BALB/c
Compared with CRISPR-Pro-based techniques, TurboKnockout® is free of patent disputes, provides precise models on a comparable timeline, and is the technology of choice among our clients performing drug development projects.
Please see below for supporting information related to Cyagen animal orders.
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