10] Suicide selection of mammalian cell mutants

Abstract

Publisher Summary This chapter describes suicide selection of mammalian cell mutants. The principle of suicide selection of mutant cells was used in microbial genetics prior to its application to mammalian somatic cells. In this procedure, a population of cells, most of which display wild-type growth characteristics and a few rare ones with additional mutant nutritional requirements, is placed in a selective medium in which the former but not the latter can grow. An agent that will kill growing cells is then added. Bromodeoxyuridine- (BrdUrd-) visible light used as the killing agent to isolate different mutants. The chapter describes the standard “BrdUrd-light” protocol and discusses the parameters that may require adjustment for particular cell types and applications. This procedure depends on the differential incorporation of the thymidine analog, bromodeoxyuridine (BrdUrd), into the DNA of growing but not into nongrowing cells in selective conditions sufficient for growth of wildtype but not mutant cells. After incorporation of BrdUrd, the cell culture is exposed to “visible light,” a component of which in the range of 313 nm results in photolysis of BrdUrd-containing DNA and death of wild-type (growing) but not mutant (nongrowing) cells.