ES Cell-Based Mouse Models

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, we will generate a customized model with a specifically modified gene in one of our proprietary high performing embryonic stem (ES) cell strains.
– ingenious is where quality meets value. With nearly two decades of experience and many benefits in place, full service projects with ingenious provide the most reliable and economic means of obtaining the model you need.

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

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 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.
  • Cost effective utilizing our proprietary technologies.

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.