18] Toxin — antitoxin selection for isolating heterokaryons and cell hybrids

Abstract

Publisher Summary This chapter discusses the methods used in toxin–antitoxin selection for isolating heterokaryons and cell hybrids. The behaviors of cell hybrids have employed genetic selection techniques, such as the HAT system, to isolate cell fusion products. Some “half-selective” strategies are devised where an established immortal cell line is fused to a normal diploid cell type which does not divide in culture, in which it is unnecessary to select against the non dividing normal diploid parental cell type. However, the inability to use genetic selection techniques with normal cells frequently represents a major limitation for many types of studies. Two alternative systems for isolating heterokaryons and cell hybrids developed are discussed in the chapter. The first one exploits the different specificities of two irreversible biochemical inhibitors. Iodoacetamide is directed primarily toward sulfhydryl groups, while diethyl pyrocarbonate reacts mainly with histidine residues. An elegant technique for introducing macromolecules into cells is introduced. Cells are first exposed to a hypenonic sucrose solution containing the molecule. Normally, the pinocytic vesicles that are formed during this period would be transported to the lysosomes, where their contents would be degraded.