Original reference: Bradley lab ES cell protocols courtesy of Guangbin Luo.

The most fundamental criteria for doing injections are having a cell line with an extensively characterized targeted event--both sides of the event evaluated with external probes or multiple overlapping internal probes. The cell line should be euploid and mycoplasma negative.

-Grow the cells in your regular ES cell culture medium without selective agents like G418. These agents may be toxic to embryos. Thaw and culture your ES cells as you normally would, on or off of feeders as appropriate. Generally two days is sufficient to have a near confluent 35 mm plate ready for passaging.

-For injection we will need two wells of a 24-well dish that are sub-confluent and off of feeders. We recommend preparing them as follows:

-On the day before the injection, prepare a 24-well dish by applying to each of 6 wells 0.5 ml of sterile 0.1% gelatin. Aspirate the gelatin off and allow the plate to dry in the hood with the fan on for approximately 30 minutes.

-Trypsinize your ES cells as you would for normal passaging from the 35mm plate. Use 1.0 ml tryspsin.

-Put the cells into 1.0 ml of your regular ES cell culture medium supplemented with LIF.

-Mix the cells thoroughly and remove 200 µl, 100µl and 50µl aliquots in duplicate and place them in the wells of the 24 well plate, prepared above, containing 1.0 ml of medium supplemented with LIF, per well. Mix each well thoroughly with a P1000. The rest of the cells may be frozen down or passaged as you see fit.

-The next day feed the wells with 0.5 ml medium and transport the plate down to the Wolstein security desk (call from the desk and weÕll come up). This will generally be on a Thursday.

-We will process the cells from there. The duplicates provide a back up should we need to redo the cell prep and the higher dilutions will provide cells for Friday.