Gene Targeting in Embryonic Stem Cells

ES CellsThis procedure includes the electroporation of one vector into one ES cell line; selection of electroporated cells; MEF cell expansion for ES cell support; picking of up to 384 ES colonies when positive selection is used (up to 192 when positive and negative selection are used); preparing of confluent 96 well plates for investigator analysis; cryopreservation of duplicate 96 well plates and a single vial for each colony; thawing, expansion and chromosome counting of up to 3 homologous recombinant clones. ES cell lines available for transfection: TC1 (129); CJ7 (129); JM8.A3 (B6/N). Additional charges are applied for supplemental services (please inquire). Please consult with the Core Facility Director prior to generating your targeting construct. Demonstration of a successful screening strategy to identify homologous recombinants is required prior to electroporation.

 

General Overview of the Process

  1. Consult with the core facility director regarding construct design.
  2. Generate construct and develop screening assay.
  3. Purify construct and provide documentation of optimized screening assay.
  4. Electroporation of ES cell colonies, expansion to replicate plates.
  5. Picking ES cell colonies, expansion to replicate plates.
  6. Screening for recombinant clones.
  7. Expansion and chromosome counting of recombinant clones.
  8. Confirmation of genotype of recombinant clones.
  9. Proceed to blastocyst injection or in vitro differentiation of targeted ES cell.
  10. If chimeric mice were made, screening for germline transmission.
  11. Breeding of heterozygote mice together to generate mice homozygous for the introduced genetic modification.

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