Detailed Project Information

Gene Targeting

Gene Targeting Projects are conducted in two phases:

Phase I: ES Cell Targeting
In Phase I, targeting vector DNA is electroporated into ES cells, the electroporated cells are then grown in drug selection media, and surviving colonies are picked into 96 well plates. Colonies growing in the 96-well plates are divided into replica plates: one set is frozen and stored at 80oC for future use, the second set is provided to the investigator for DNA isolation and genotyping.

Phase II: Production of Chimeric Mice
In Phase II, potentially targeted clones generated in Phase I are thawed from the 96 well plates, expanded, and frozen for stable long-term storage in LN2. Expanded clones confirmed as correctly targeted by the investigator are scheduled for blastocyst microinjection. Targeted ES clones are microinjected into blastocysts to generate chimeric mice.

Gene Targeting Project Initiation

Information you need to provide to initiate a project:

• Investigators must have an NUACUC approved animal protocol describing the use of animals generated by the TTML before microinjections can be scheduled.
• Investigators from CMRC must also provide a copy of their current Institutional Biosafety Committee approval letter
• A completed Gene Targeting request form submitted to TTML@northwestern.edu (Word)
• Purified electroporation DNA: ~100µg total, linearized, purified DNA prepared at a concentration of at least 2µg/ul sterile TE
• Evidence of a robust screening assay for analysis of homologous recombination in selected clones. It is incumbent up on the investigator to develop a sensitive screening assay and to demonstrate the ability of probes to detect control (wild-type) DNA in 2-5 µg genomic DNA isolated from a 96 well plate (Protocol). 96 well practice plates are available upon request. (Request practice plates)
• TTML staff provides guidance and advice on targeting vector design, screening strategies, and data interpretation.

Once the completed request form and purified DNA is received, we will:

• Review your screening assay
• Verify DNA concentration by fluorometry and assess targeting vector integrity by gel electrophoresis; DNA of inadequate concentration or purity will be returned
• Schedule electroporation