Full Service

Service Steps from GeneTargeting.comIf you can think it up, we can knock it out (or in)! From concept to experimental reality, ingenious has delivered mouse models that have advanced the research of over 900 laboratories worldwide. Based on your requirements, ingenious will generate a customized mouse model with a specifically modified gene in one of our proprietary high performing embryonic stem (ES) cell strains or via CRISPR injection. With over 16 years of experience in both the pharmaceutical industry and academia, full service projects with ingenious are the most reliable and economic approach to creating genetically modified mouse models. This means we generate your mouse model from design of the targeting vector to delivery of germline confirmed mice. This includes, but is not limited to the full scope of target modification to generate Constitutive or Conditional Knockout Mouse Models, Knockin Mouse Models, Truhumanization Mouse Models, Inducible/Reversible Mouse Models (F.A.S.T ™), and Targeted Transgenic Mouse Models. Utilizing homologous recombination in ES cells or CRISPR Cas9 genome editing combined with proprietary technologies results in the production of sophisticated, next generation mouse models.

 

ES cell based mouse models

ES cell based mouse models are the gold standard for the most accurate and reliable models. From the moment you contact one of our scientific professionals at ingenious, a thorough assessment of the risks and benefits for the mouse model that yuo wish to generate along with an accurate timeline as well as costs will be provided. We take the time to outline all potential options so you can make an educated decision on which strategy might best fit your needs.

When working with ingenious, the following service steps are performed:

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Project Evaluation and Initiation

Together we carefully evaluate your needs and research goals and translate them into personalized project parameters for your particular mouse model.
Before a project is initiated, the gene of interest is thoroughly evaluated at no extra cost, and all potential strategies are discussed with you to determine which strategy would best fit your experimental objectives.

Vector Creation

  • Complete bioinformatics analysis of gene structure and sequence content.

  • Technical review of any previous targeting history of the gene of interest.

  • Presentation of all relevant targeting strategies that meet your experimental objectives.

  • Screening strategy design for PCR and Southern blot analysis.

  • Confirmation of genomic BAC clone containing the target gene.

  • Cloning and confirmation of homology arms.

  • Insertion and confirmation of the Neomycin selection cassette flanked by FRT and/or loxP sites.

  • Engineering/confirmation of specific mutations/deletions/insertions according to the project specifics.

  • Sequencing of targeting vector.

  • Linearization of DNA for electroporation.

Electroporation and Tissue Culture

  • Culture and expansion of ES cells.

  • Electroporation of ES cells with targeting vector.

  • Positive/negative selection of ES cells.

  • Isolation of 200-300 ES cells per electroporation.

  • Duplication and freezing of ES cells.

  • Isolation of DNA for screening of ES cells.

  • Click here to learn more about our ES Cell Lines.

Screening

  • PCR and Southern blotting for identification and confirmation of positive clones.

  • Genomic stability of ES cell clones is assessed by determining the percentage of euploid cells to ensure that ES cells of high genomic stability are used for injection and mouse production.

  • Expansion of positive ES cell clones and preparation for microinjection.

Injection

  • Preparation of embryos and foster mice.

  • Microinjection of targeted ES clones into blastocysts or laser assisted injection of 8-cell stage embryos.

  • Implantation of blastocysts/8-cell embryos.

Animal Husbandry and Confirmation of Germline Transmission

  • Maintenance and care of recipient females and subsequent weaning of chimeras.

  • Breeding of male chimeras with C57Bl/6N wild type females.

  • Weaning of the F1 generation.

  • F1 Genotyping via tail biopsy and DNA extraction.

  • PCR analysis and confirmation of project specific modifications as well as Neo deletion and absence of FLP transgene.

After You Receive Your Mice

Full projects with ingenious take you from concept to mice and our post project support services take you the rest of the way. Our team of experts, utilizing proprietary prediction and modeling software, can generate the cohorts you need for your experiments within timelines and cost that best fit your lab. Our cryopreservation services help you protect your investment in generating your mouse lines.

CRISPR-based mouse models

CRISPR gene editing is an exciting technology that clients can utilize to generate mouse models for their research. For your custom project, we can use CRISPR by microinjection or via CRISPR-Assisted technology in ES cells, depending on your scientific parameters. Please contact one of our scientific professionals to determine which technology is the most appropriate strategy for your custom model.

CRISPR via ES cells

Simple conventional knockouts and point mutation knockins can be generated by utilizing CRISPR assisted homologous recombination in ES cells.

  1. Project strategy consultation

  2. Design and preparation of reagents and donor targeting vector based on project specifications

  3. Electroporation and tissue culture using FLP ES cells

  4. Screening of ES cells via PCR to identify positives

  5. Microinjection into blastocysts for production of chimeras

  6. Mating of chimeras to obtain germline-confirmed F1 mice