Protocols

Preparation of ES Cells for Blastocyst Injection

Actively growing but not yet confluent ES cells from one 60 mm dish must be provided no later than the time requested by core personnel on the mornings scheduled for blastocyst injection.

Targeted ES cells are thawed and expanded on MEFs prior to microinjection. The cells are usually grown on T-25s or P60s.

Approximately 5 days before injection:

Bring up cells to be injected on a T-25 flask or other appropriate surface.

Cells will be moved to three p60 dishes with varying concentrations (0.5 x 106, 1.0 x 106, and 1.5 x 106) to give a choice of optimum plates on injection day.

Injection Day:

Feed cells with fresh cES cell media 2 to 3 hours prior to trypsinization.

1 hour before drop-off time:

Choose the plate with the optimum cells and rinse with 5 ml PBS two times.

Add 1 ml trypsin.

Incubate at 37°C for 3 to 5 minutes.

Add 4 ml cES cell media.

Pipette up and down until colonies are broken into a single cell suspension.

Plate single suspension cells onto two 60 mm pre-gleatinied dishes with 3 mls each cES for 45 to 60 minutes.

After a fair amount of cells have settled on the bottom, gently aspirate non-adherent cells with a 10 ml pipette. Save this supernatant in a 15 ml conical tube at 4°C as a last resort back-up.

With 2 ml of fresh cES cell media, 'blow off'' loosely adhering ES cells with pipette and transfer to a 15 ml conical tube. Check for single cell suspension by looking at a drop of media/cells under microscope. Continue pipetting up and down to break into a single cell suspension if necessary.

Bring the cells in a 15 ml conical tube on ice to the Vanderbilt Transgenic Mouse / Embryonic Stem Cell Shared Resource. Also bring a separate tube containing 15 ml of ES cell media with serum.