TurboKnockout® Knockout & Conditional KO Mice

Knockout and Conditional Knockout Mice | Cyagen

 

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.

>> Learn more about TurboKnockout

 

Cyagen TurboKnockout® Knockout (KO) and Conditional KO (cKO) Mice

Conventional knockout (KO) Mouse Models

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

 Knockout Mice - Conventional/Constitutive Knockout (KO) Strategy | Cyagen

 

Conditional Knockout (cKO) Mouse Models

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.

  • Gene Function Studies: Inducible and/or tissue-specific gene expression – knockout, over-expression, etc.
  • Target Discovery and Confirmation: cKO models solve the problem of embryonic lethality seen in KO models, enabling target confirmation in adult organisms.
  • Verifying Compound Specificity: cKO models allow precise control of gene expression for a functional protein target, providing an in vivo system to evaluate potential off-target effects of a drug.

 

Using Cre-Lox Recombination – Gene Floxing

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

 Conditional Knockout Mice - Gene Floxing (Cre-LoxP) Strategy Example | Cyagen

 

Knockout-First Conditional Allele Strategy

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

 Conditional Knockout Mice - Knockout-First (KO-First) Strategy Example | Cyagen

 

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). 

 

Workflow for TurboKnockout® Knockout (KO) and Conditional KO (cKO) Mice

TurboKnockout Humanized & Knockin Mouse Models | Cyagen

Traditional ES Humanized and Knockin Mice | Cyagen

1. TurboKnockout® gene 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. TurboKnockout® 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 embryonic stem (ES) cells

  • Electroporate targeting vector into our super competent TurboKnockout® ES cells, followed by appropriate drug selection and isolation of drug-resistant clones. By default, our ES cells are derived from the C57BL/6 strain. Additionally, we offer services using BALB/c-derived ES cells.
  • Screen for targeted ES cell clones by PCR.
  • Expand up to six PCR-positive clones, which are further confirmed by Southern blot.
  • Karyotype ES cells to ensure correct chromosome number.

 

4. TurboKnockout® mouse production

  • Introduce targeted ES cells into host embryos, followed by transfer into surrogate mothers. The resulting TurboKnockout® heterozygous mutants are 100% derived from the injected ES cells. We will aim to generate a minimum of 3 mice.
  • Genotype founders by PCR to confirm their mutant status.
  • Optional: Breed to F1.
  • Delivery of mice to the customer

 

Pricing and Turnaround Time

Service Price (Standard B6 strains) Turnaround time
TurboKnockout® targeting strategy design Free 1-4 days
Construction of TurboKnockout® gene targeting vector $8,950 8-14 weeks
Gene targeting in ES cells $12,950 8-14 weeks
Karyotype ES cells to ensure correct chromosome number $350/clone 1-2 weeks
TurboKnockout® mouse production $8,950
(we will inject as many ES cell clones as needed to fulfill our guarantee)
8-12 weeks
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.

 

Donor Strain Information

Mouse: C57BL/6, BALB/c

 

Advantages of TurboKnockout® Humanized & Knockin Mice

Compared with CRISPR/Cas9-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.

  • Freedom to Operate: Technology of choice for drug development projects
  • Success Rate: 100% guaranteed germline transmission (GLT)
  • Turnaround Time: Founders as fast as 6-8 months
  • Large Fragment Knockin: Insert fragments up to 300 kb
  • Flexible Floxing: No size limitations for your floxed region
  • 100% Money-Back Guarantee

 

Order Supporting Information 

Please see below for supporting information related to Cyagen animal orders.
https://www.cyagen.com/us/en/support/order-information.html

 

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.

 

Why work with Cyagen?

  • 14+ years' experience in custom animal model generation
  • 78,000+ animal models generated and delivered worldwide
  • 4,700+ citations in SCI journals for our services
  • 3,000+ university and company partners
  • 100% money-back guarantee option available for most services
  • AAALAC-Accredited and OLAW Assured