Generation of Multiple Knockout Mutants Using the Cre-loxP System

Dictyostelium discoideum is an exceptionally powerful system by which to study various aspects of modern cell and developmental biology. The completion of the genome sequencing project together with a high-efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, as a result of many gene products with overlapping functions, there is great need to produce mutants carrying mutations in multiple genes. We have, therefore, developed a robust system and describe a protocol for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of Cre recombinase.