title: Transformation of Dictyostelium discoideum with plasmid DNA.
abstract: DNA-mediated transformation is one of the most widely used techniques to study gene function. The eukaryote Dictyostelium discoideum is amenable to numerous genetic manipulations that require insertion of foreign DNA into cells. Here we describe two commonly used methods to transform Dictyostelium cells: calcium phosphate precipitation, resulting in high copy number transformants; and electroporation, an effective technique for producing single integration events into genomic DNA. Single integrations are required for gene disruption by homologous recombination. We also discuss how different selection markers affect vector copy number in transformants and explain why blasticidin has become the preferred selectable marker for making gene knockouts. Both procedures can be accomplished in less than 2 h of hands-on time; however, the calcium phosphate precipitation method contains several incubations, including one of at least 4 h, so the total time required for the transformation is approximately 8 h.
authors.affiliation: dictyBase, Center for Genetic Medicine, Northwestern University, 676 North Saint Clair Street Suite 1260, Chicago, Illinois 60611, USA.
authors.forename: Pascale
authors.initials: P
authors.lastname: Gaudet
authors.forename: Karen E
authors.initials: KE
authors.lastname: Pilcher
authors.forename: Petra
authors.initials: P
authors.lastname: Fey
authors.forename: Rex L
authors.initials: RL
authors.lastname: Chisholm
mesh_terms: D000818:Animals; D002130:Calcium Phosphates / Q000494:pharmacology*; D004247:DNA / Q000235:genetics*; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D017353:Gene Deletion; D005819:Genetic Markers; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics*; D013997:Time Factors; D014170:Transformation, Genetic
chemical_list: D002130:Calcium Phosphates; D005819:Genetic Markers; D004247:DNA; C020243:calcium phosphate