ES Cell Targeting

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 (see protocol) and analysis of genomic DNA.

Screening results are expected from the investigator within two 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 two months.

• 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

Investigators are responsible for the following in 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 two electroporations per targeting construct, even if provided DNA is toxic as compared to controls, resulting in low numbers of drug-resistant colonies.

ES cells are grown in selection media for six to 10 days following electroporation. Approximately 200 to 250 clones that survive selection, and have therefore integrated the targeting vector DNA, will be provided in a 96 well plate format. You will need to isolate DNA from each well and assess the DNA to determine whether the targeting vector integrated randomly or via homologous recombination.

Following drug selection, colonies are picked into a 96 well plate and allowed to grow for several days. When confluent, the cells in each 96 well plate are split into four replica 96 well plates. Two plates are frozen and stored at –80°C for future use. The other two are placed back into the incubator and cells continue to grow until wells are slightly overgrown. Wells are then washed with PBS, frozen, and provided for DNA isolation and analysis.

We recommend isolating DNA from one replica plate initially. Expect to isolate enough DNA from each well for a PCR reaction and one restriction digest. If precipitated DNA is resuspended in 40µl/well of buffer, 1µl is used for PCR and 30µl for a Southern leaving a bit of DNA to spare. All wells can be screened initially by PCR for homologous recombination at one arm. PCR positive clones are then screened by Southern for recombination at the remaining arm. The second 96 well plate is used for a second Southern (if Southerns are used to analyze both ends) or as a back-up.

ES cell clones initially frozen in the 96 well plates are stored at –80°C during the screening period. Cells kept under these conditions are not stable over long periods of time and should optimally be thawed within two months. This is one of the reasons it is so important to have a well-defined, robust screening assay prior to electroporation.

Potentially targeted clones identified in the initial screen are thawed, expanded, frozen and stored under stable conditions in LN2 dewars. It is important that these clones are confirmed by Southern as correctly targeted at both the 5' and 3' ends prior to microinjection. This ensures that the correct clones have been thawed and are of interest. It would be unfortunate to discover 6 months down the road that the mice you have been working with do not have the expected mutation. Additional DNA from the thawed/expanded clone(s) will be provided at this step.