Gene Targeting in Embryonic Stem Cells
This 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
- Consult with the core facility director regarding construct design.
- Generate construct and develop screening assay.
- Purify construct and provide documentation of optimized screening assay.
- Electroporation of ES cell colonies, expansion to replicate plates.
- Picking ES cell colonies, expansion to replicate plates.
- Screening for recombinant clones.
- Expansion and chromosome counting of recombinant clones.
- Confirmation of genotype of recombinant clones.
- Proceed to blastocyst injection or in vitro differentiation of targeted ES cell.
- If chimeric mice were made, screening for germline transmission.
- Breeding of heterozygote mice together to generate mice homozygous for the introduced genetic modification.