MEF feeders are prepared weekly to provide a substrate for undifferentiated embryonic stem (ES) cells. Primary MEF cells are thawed, established in culture, treated with mitomycin C to halt their proliferation so they cannot overgrow the ES cultures, and then replated onto dishes convenient for ES cell culture. This protocol can also be used to prepare feeder cells from STO fibroblast cell lines. - [Read Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates Protocol]
The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.