Original reference: "Tissue Culture made Easy" by Christian LaMantia from the Magnuson lab. Preplating protocol comes from the Doetschman chapter in Transgenic Animal Technology: a Laboratory Handbook, ed. Carl Pinkert (1994).
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. Some injection facilities also require that you test for mycoplasma. One derivatized cell-line from this facility was determined to be mycoplasma negative by ATCC (a copy of the results is in the protocol book).
The goal will be trypsinized single cells (not clumps), but cells not trypsinized for so long that they've become sticky. This is achieved by doing the digestion at room temperature under the micro-scope for approximately 6 minutes. Have a backup plate in case of problems since injections are precious.
-Grow the cells in ES cell culture medium without antibiotics or selective agents like G418. These agents may be toxic to embryos.
-24 hours before the scheduled injections passage recently thawed targeted cell lines to generate a sub-confluent 35 mm dish. This will ensure that the cells are viable.
-Wash the plate a couple of times with culture medium to remove any floating cells, then cover dish with freshly thawed trypsin. Remove this trypsin and apply new trypsin.
-Put the dish on the microscope and observe the digestion periodically. When ready, almost all of the ES cells within each colony will look rounded. This is the most important step because if you under-trypsinze the cells they will be clumpy and if you over-trypsinize they will be sticky, therefore hard to inject. This normally takes about 6 minutes for cells grown exclusively on gelatinized plastic. Lines that grow on feeders may take a little longer. A good indicator is that the cells should rinse off easily when you go to break up the clumps.
-Break the colonies up with a plugged pasteur pipet (or other small bore pipet) and place the cells in an equal volume of ES cell culture medium.
-Spin down as usual and gently resuspend cells in 2 ml of ES cell medium. Feeders are removed by plating the cells onto a fresh gelatinized 35 mm dish and incubating for 30 min. Gently remove the supernatant and plate again on a fresh gelatinized 35 mm dish. Incubate for 1 hour. The fibroblasts should now be largely attached. Gently remove supernatant and transfer to a convenient size tube. Place tube on ice and deliver to the injection facility.
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