KOMP: High-throughput production

Production of mice from ES cells

The first phase of the KOMP program focused on generating targeted knockout mutations in mouse ES cells. The second phase, KOMP2, relies upon the successful generation of strains of knockout mice from these ES cells.  Our KOMP2 Production Center will draw upon the 80-plus years of experience at The Jackson Laboratory (JAX) in delivering top-quality models to the scientific community to produce germline-competent chimeras and subsequent mouse cohorts. 

The JAX KOMP2 Production Center will:
  • Produce and assure the genetic quality of 833 mouse knockout strains utilizing ES cells from IKMC resources; 
  • Evaluate the viability and fecundity of each homozygous strain and analyze LacZ expression patterns in heterozygous mice, depositing the relevant data in the KOMP2 Data Coordination Center Database (DCCDB); 
  • and Breed and distribute cohorts of knockout animals to KOMP2 Phenotyping programs for comprehensive gene function studies, as well as depositing cryopreserved germplasm with the KOMP Repository. 

Production pipeline

Based on JAX’s strengths in mouse production and distribution capacity, large microinjection infrastructure, high-throughput cryopreservation and assisted reproductive technologies, we have developed a production pipeline to introduce new technologies to improve capacity and success. 

The JAX KOMP2 production pipeline outlined below comprises several key components to ensure the quality of the cohorts sent for phenotyping, to provide information regarding gene expression, viability and fecundity, and to increase the efficiency of mouse production, ultimately reducing the costs and time required to create knockout mice.

Importation and expansion of ES cells from IKMC partners.  These include both “knockout-first” (from KOMP and EUCOMM) and full gene replacement (KOMP) alleles targeted to C57BL/6N ES cell lines. Visit the IKMC web site for more information about the targeting strategies and allele types. 

Genetic quality control. Prior to production of cohorts, it is essential to confirm the proper targeting of each allele. JAX has adopted a rigorous screening process to confirm targeting in both ES cells and mice, and to provide genotyping assay information to the scientific community. 

ES cell culture and microinjection. All ES cells are microinjected into B6(Cg)-TyrC-2J/J (Stock #000058) blastocysts to generate chimeras. JAX has been developing optimized culture conditions to improve chimera generation rates and germline transmission rates. ES cell clones are also screened for chromosome number to ensure injection of euploid cells. 

Chimera breeding and germline transmission. Chimeras are bred to C57BL/6NJ (Stock #005304) to ensure isogenicity of the progeny with the ES cell strain background. 

Cre-mediated deletion. All KOMP2 mice must be bred to a Cre deleter strain to remove 1) the neo cassette harboring a heterologous promoter and 2) create a “definitive null” in knockout first alleles by eliminating the critical loxP-flanked exon. JAX has generated a new C57BL/6NJ version of the efficient Sox2-Cre driver (Stock# 014094) to accomplish this goal.

LacZ expression. Using the pipeline we developed for the JAX Cre Repository, we are examining expression patterns of the reporter allele in adult animals (male and female) and E12.5 embryos. All expression data (annotations and images) will be available through the KOMP2 DCCDB.

Viability/embryonic lethality. At least one-third of all homozygous knockouts are expected to be embryonic lethal. An initial screen will establish whether lethal mutants are present and if they harbor overt phenotypes at E12.5 and at E18.5, capturing basic information for more extensive subsequent analysis. 

Fertility screening. Based on the experience of the JAX ReproGenomics program, we expect approximately 10% of knockout strains to be infertile. We are screening KOMP2 mice for this phenotype, determining if the phenotype is female or male (or both) infertility, and performing a basic histological analysis of reproductive organs of infertile strains. 

Cohort breeding. Cohorts of 8 male and 8 female knockout mice are being generated for the KOMP2 Phenotyping Program. For subviable and lethal strains, cohorts of heterozygous mice will be sent for phenotyping.  

Cryopreservation. All strains, including the knockout-first “native” allele and definitive knockout allele will be cryopreserved to generate an ongoing resource for the scientific community.