General protocol for microcell-mediated chromosome transfer

We have developed a general technique for making micronucleated cells to use in microcell-mediated chromosome transfer. Growing cells are blocked in mitosis with colcemid, placed in a hypotonic solution for 10 min, and returned to culture medium for 24 h. This treatment promotes the formation of micronuclei within lymphoblast or fibroblast cells. The microcells are generated by cytochalasin B treatment on a Percoll density gradient centrifuged at 43,500g. The resulting mixture of microcells, whole cells, and karyoplasts is filtered through 3-micron pores to obtain a pure microcell preparation. The microcells are fused to recipient whole cells using phytohemagglutinin-P and polyethylene glycol. Advantages of this technique are: donor cells need not be attached to a substrate; and cell lines which form micronuclei in low frequency can still be used efficiently as microcell donors.