Transformation of dictyostelium with plasmid DNA by calcium phosphate precipitation.

Abstract

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Calcium phosphate precipitation is a commonly used method for DNA-mediated transformation in Dictyostelium. Calcium phosphate precipitation produces high-copy-number transformants and is often used for overexpression experiments in conjunction with the G418 resistance gene, which needs to be present at high levels to produce efficient selection.