Transformation of Dictyostelium discoideum with plasmid DNA

Pascale Gaudet, Karen E. Pilcher, Petra Fey, Rex L. Chisholm*

*Corresponding author for this work

Research output: Contribution to journalArticle

68 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1317-1324
Number of pages8
JournalNature Protocols
Volume2
Issue number6
DOIs
StatePublished - Jun 1 2007

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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