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 -80°C for future use, the second set is provided to the investigator for DNA isolation and genotyping.
Generation of targeted clones
- ES cells are electroporated with the targeting vector and placed under drug selection. Approximately 200-250 drug-resistant colonies are picked into 96-well plates that are frozen and stored short-term at -80°C. Two additional replica plates are provided to the investigator for isolation (Protocol) and analysis of genomic DNA.
- Screening results are expected from the investigator within 2 months (why 2 months?). Review of initial screening results with the investigator is required before potentially targeted clones are expanded.
- For most projects, targeted clones are identified in the first set of provided plates. However, if targeted clones are not identified, another electroporation will be scheduled and additional clones (up to a total of 480) will be provided for the same fee. Consultation is required prior to the second electroporation.
- Gene targeting via homologous recombination (targeting) is highly dependent on the specific locus being modified, and, therefore, we cannot guarantee that targeting will occur with a particular construct.
- Projects are limited to 2 electroporations per targeting construct. Redesign of the targeting vector is strongly recommended if no targeted clones are identified following 2 electroporations (up to 480 clones screened). Electroporation DNA that is toxic, as compared to controls, may result in the generation of significantly fewer selectable colonies.
- Germline is not guaranteed for clones stored in 96 well plates for longer than 2 months (why?).
Gene targeting timeline
- Week 1: set up for electroporation
- Week 2: electroporation and begin selection
- Week 3: pick colonies into 96 well plates
- Week 4: freeze replica 96 well plates/set up replica 96 well DNA plates
- Week 5: 96 well plates provided to lab for DNA isolation/analysis
Investigator responsibilities for gene targeting projects:
- Design and construction of targeting vector
- Perfecting a robust screening assay that will detect a targeted homologous recombination event in ES cell clones
- Proficiency at isolating small amounts of genomic DNA from individual wells of a 96-well plate
- Preparation of linearized endotoxin-free electroporation quality targeting DNA for electroporation. Projects are limited to 2 electroporations per targeting construct, even if provided DNA is toxic as compared to controls, resulting in low numbers of drug-resistant colonies.
Frequently Asked Questions
- How are clones provided following electroporation?
- Why did I receive two sets of 96 well plates for screening?
- How much time do I have to complete the initial 96-well plate screen?
- Why do I need to confirm targeting?
Costs and Services
See Gene Targeting costs and services