Protocol

Selection of Dictyostelium Transformants

dictyBase, Center for Genetic Medicine, Northwestern University, Chicago, IL 60611, USA

¹Corresponding author (pgaudet{at}northwestern.edu)

INTRODUCTION

Dictyostelium 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. Two commonly used methods for DNA-mediated transformation in Dictyostelium are calcium phosphate precipitation and electroporation. Transformants can then be selected in liquid media or on bacterial plates. The latter method reduces the chances of contamination because the cells are grown in buffered agar containing live or dead bacteria, rather than in a rich broth. This method also facilitates the isolation of clones from transformations because each transformant produces a single colony on the plate instead of the pools of transformants obtained in liquid culture. For gene ablation experiments, it is important to obtain a minimum of two independent clones with the same phenotype to exclude the possibility that the phenotype is due to a nonspecific mutation.