It is critical to have the cells in log phase growth for microinjecting to produce germline transmission chimeric mice. Confluence should be ~60-70%, with many small to medium size colonies. The colonies should be small, tight, have smooth raised edges. The overgrown ES cells will reduce their pluripotential and do not contribute as large a percentage to the embryo. The result is lower numbers of chimeric animals and lower percentage of chimerism in the individual founders with reduced germline transmission.
ES cells used for injection can be grown on 12 well or 6 well plate. Due to individual frozen vials have a different number of viable cells; it is wisely to seed cells at three dilutions of each clone for injection. A simple method is to thaw your vial of cells for injection, or passage the clone, seed 1/2 in one well, 1/3 in the second well and 1/6 in the third well. After two to three days, one of the well will be at sub-confluence and right for microinjection.
Procedure:
- Feed ES cells 1-2 hours prior to trypsinization.
- Rinse plate twice with DPBS.
- Trypsinize cells with 0.05% Trypsin/EDTA for 2-4 minutes at 37oC.
- Disperse ES cells with ES Media to obtain a single cell suspension.
- Spin cells down and re-suspend the cells in 4 ml of ES medium.
- Plate onto one 60 mm gelatinized dish for 1 hour.
- After 1 hour, aspirate non-adherent cells.
- With 2 ml of fresh media, wash off loosely adhering ES cells with pipette and
- Spin cells down and re-suspend the cells in ice cold M2 Medium, and leave on ice.
|
