When genetically altered ES cell lines are identified, thaw the 96-well plates, or cells from vials, onto feeders, and grow them up. Avoid taking them through too many passages, since with increasing passage number, the possibility of cells loosing their germline-transmitting ability may increase. Our standard protocol for preparing cells for aggregation is given below. Preparation of cells for blastocyst injection is performed in a similar manner, except that for this procedure, the cells need to be seeded at the usual density on d 3, trypsinized slightly longer, spun down and washed in PBS to obtain single-cell suspension on d 5 (below).
Day 1: Thaw cells 4 d prior to aggregation on a feeder cell layer containing plate. Day 2: Change the medium.
Day 3: Split cells onto gelatinized plates, but instead of the usual 1:5 ratio, pass them 1:50 or even more dilute (see Notes 21 and 22). Day 4: Change the medium.
Day 5: Trypsinize the cells briefly, until the colonies lift up as a loosely connected clump of cells. Stop trypsin by adding DMEM+ to the plate. Select clumps directly form the plate for aggregation (see Note 23).
Protocols and descriptions detailing the introduction of cells into mice by aggregation of ES cells with preimplantation embryos are provided elsewhere (40). Our own lab protocols can be obtained through the World Wide Web at http://www.mshri.on.ca/develop/nagy/nagy.htm.
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