ES Cell Preparation for Blastocyst Injections

Help with designing your targeting vector and embryonic stem cell transfection service is offered by the Siteman Cancer Center Stem Cell Core. The MGC will coordinate arrangements with the Stem Cell Core to prepare and transfer your targeted cells on injection days. If you provide your own cells, please use the following protocol to prepare them for injection:

Thaw cells from appropriate positive clone or clones onto a 6cm dish plated with a MEF feeder layer at least 3-5 days prior to injection dates depending on growth speed of your cells. Split cells just before they become confluent, usually 2-3 days before injections, onto three 6cm plates – one for each day of injections as follows: 50% for injection day 1; 30% for day 2; and 20% for day 3.

On the morning of the injections, feed the cells two hours prior to injection time. Just before the scheduled time of transfer, trypsinize the cells for five minutes with pre-warmed trypsin at 37ºC. Place the trypsinized cells in a 15 mL tube, and add an equal volume of ES cell media to inactivate the trypsin. Centrifuge the cells 100-200 x g and remove the supernatant. Resuspend the cells in 1mL of 'injection media' (see below) + 10% FBS. Prepare a second tube containing 10mL injection media + 10% FBS for use on our injection slides. Complete separation of ES cells is essential for blastocyst injections - please GENTLY pipette cells 30-40 times to ensure cell separation.

Bring the cell suspension and extra injection media to us on wet ice.

Injection Media: (available through the tissue culture center on campus)

DME
25mM HEPES without NaHCO3
Osmolarity = 290
PH = 7.4

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