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Transformation of Dictyostelium discoideum with plasmid DNA

Nature Protocols volume 2, pages 1317–1324 (2007)Cite this article

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.

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References

  1. Manstein, D.J., Titus, M.A., De Lozanne, A. & Spudich, J.A. Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J. 8, 923–932 (1989).

    Article  CAS  Google Scholar 

  2. Simon, M.N., Driscoll, D., Mutzel, R., Part, D., Williams, J. & Veron, M. Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum . EMBO J. 8, 2039–2043 (1989).

    Article  CAS  Google Scholar 

  3. Crowley, T.E., Nellen, W., Gomer, R.H. & Firtel, R.A. Phenocopy of discoidin I-minus mutants by antisense transformation in Dictyostelium . Cell 43, 633–641 (1985).

    Article  CAS  Google Scholar 

  4. Martens, H., Novotny, J., Oberstrass, J., Steck, T.L., Postlethwait, P. & Nellen, W. RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase. Mol. Biol. Cell 13, 445–453 (2002).

    Article  CAS  Google Scholar 

  5. Kuspa, A. Restriction enzyme-mediated integration (REMI) mutagenesis. Methods Mol. Biol. 346, 201–209 (2006).

    CAS  PubMed  Google Scholar 

  6. Nellen, W., Silan, C. & Firtel, R.A. DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion. Mol. Cell. Biol. 4, 2890–2898 (1984).

    Article  CAS  Google Scholar 

  7. Howard, P.K., Ahern, K.G. & Firtel, R.A. Establishment of a transient expression system for Dictyostelium discoideum . Nucleic Acids Res. 16, 2613–2623 (1988).

    Article  CAS  Google Scholar 

  8. Pang, K.M., Lynes, M.A. & Knecht, D.A. Variables controlling the expression level of exogenous genes in Dictyostelium . Plasmid 41, 187–197 (1999).

    Article  CAS  Google Scholar 

  9. Lloyd, M.M., Ceccarelli, A. & Williams, J.G. Establishment of conditions for the transformation of nonaxenic Dictyostelium strains. Dev. Genet. 11, 391–395 (1990).

    Article  CAS  Google Scholar 

  10. Wetterauer, B., Salger, K., Demel, P. & Koop, H. Efficient transformation of Dictyostelium discoideum with a particle inflow gun. Biochim. Biophys. Acta 1499, 139–143 (2000).

    Article  CAS  Google Scholar 

  11. Sutoh, K. A transformation vector for Dictyostelium discoideum with a new selectable marker bsr. Plasmid 30, 150–154 (1993).

    Article  CAS  Google Scholar 

  12. Egelhoff, T.T., Brown, S.S., Manstein, D.J. & Spudich, J.A. Hygromycin resistance as a selectable marker in Dictyostelium discoideum . Mol. Cell. Biol. 9, 1965–1968 (1989).

    Article  CAS  Google Scholar 

  13. Chang, A.C., Hall, R.M. & Williams, K.L. Bleomycin resistance as a selectable marker for transformation of the eukaryote, Dictyostelium discoideum . Gene 107, 165–170 (1991).

    Article  CAS  Google Scholar 

  14. Leiting, B. & Noegel, A.A. The ble gene of Streptoalloteichus hindustanus as a new selectable marker for Dictyostelium discoideum confers resistance to phleomycin. Biochem. Biophys. Res. Commun. 180, 1403–1407 (1991).

    Article  CAS  Google Scholar 

  15. Chang, A.C., Williams, K.L., Williams, J.G. & Ceccarelli, A. Complementation of a Dictyostelium discoideum thymidylate synthase mutation with the mouse gene provides a new selectable marker for transformation. Nucleic Acids Res. 17, 3655–3661 (1989).

    Article  CAS  Google Scholar 

  16. Kalpaxis, D., Werner, H., Boy-Marcotte, E., Jacquet, M. & Dingermann, T. Positive selection for Dictyostelium mutants lacking uridine monophosphate synthase activity based on resistance to 5-fluoro-orotic acid. Dev. Genet. 11, 396–402 (1990).

    Article  CAS  Google Scholar 

  17. Morio, T., Adachi, H., Sutoh, K., Yanagisawa, K. & Tanaka, Y. Bsr-REMI: an improved method for gene tagging using a new vector in Dictyostelium . J. Plant Res. 108, 111–114 (1995).

    Article  CAS  Google Scholar 

  18. Barth, C., Fraser, D.J. & Fisher, P.R. Co-insertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome. Plasmid 39, 141–153 (1998).

    Article  CAS  Google Scholar 

  19. Early, A.E. & Williams, J.G. Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum . Gene 59, 99–106 (1987).

    Article  CAS  Google Scholar 

  20. Fey, P., Kowal, A.S., Gaudet, P., Pilcher, K.E. & Chisholm, R.L. Protocols for growth and development of Dictyostelium discoideum . Nat. Protoc. 2, 1307–1316 (2007).

    Article  CAS  Google Scholar 

  21. Sussman, R. & Sussman, M. Cultivation of Dictyostelium discoideum in axenic medium. Biochem. Biophys. Res. Commun. 29, 53–55 (1967).

    Article  CAS  Google Scholar 

  22. Watts, D.J. & Ashworth, J.M. Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem. J. 119, 171–174 (1970).

    Article  CAS  Google Scholar 

  23. Wetterauer, B. et al. Wild-type strains of Dictyostelium discoideum can be transformed using a novel selection cassette driven by the promoter of the ribosomal V18 gene. Plasmid 36, 169–181 (1996).

    Article  CAS  Google Scholar 

  24. Caterina, M.J., Milne, J.L. & Devreotes, P.N. Mutation of the third intracellular loop of the cAMP receptor, cAR1, of Dictyostelium yields mutants impaired in multiple signaling pathways. J. Biol. Chem. 269, 1523–1532 (1994).

    CAS  PubMed  Google Scholar 

  25. Hadwiger, J.A. & Firtel, R.A. Analysis of G alpha 4, a G-protein subunit required for multicellular development in Dictyostelium . Genes Dev. 6, 38–49 (1992).

    Article  CAS  Google Scholar 

  26. Kalpaxis, D. et al. Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid. Mol. Gen. Genet. 225, 492–500 (1991).

    Article  CAS  Google Scholar 

  27. Faix, J., Kreppel, L., Shaulsky, G., Schleicher, M. & Kimmel, A.R. A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Res. 32, e143 (2004).

    Article  Google Scholar 

  28. Betapudi, V., Shoebotham, K. & Egelhoff, T.T. Generation of double gene disruptions in Dictyostelium discoideum using a single antibiotic marker selection. Biotechniques 36, 106–112 (2004).

    Article  CAS  Google Scholar 

  29. Insall, R.H., Soede, R.D., Schaap, P. & Devreotes, P.N. Two cAMP receptors activate common signaling pathways in Dictyostelium . Mol. Biol. Cell 5, 703–711 (1994).

    Article  CAS  Google Scholar 

  30. Alibaud, L., Cosson, P. & Benghezal, M. Dictyostelium discoideum transformation by oscillating electric field electroporation. Biotechniques 35, 78–80, 82–83 (2003).

    Article  CAS  Google Scholar 

  31. Shah-Mahoney, N., Hampton, T., Vidaver, R. & Ratner, D. Blocking the ends of transforming DNA enhances gene targeting in Dictyostelium . Gene 203, 33–41 (1997).

    Article  CAS  Google Scholar 

  32. Morrison, A., Marschalek, R., Dingermann, T. & Harwood, A.J. A novel, negative selectable marker for gene disruption in Dictyostelium . Gene 202, 171–176 (1997).

    Article  CAS  Google Scholar 

  33. Sambrook, J., Fritsch, E.F. & Maniatis, T. (eds.) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

    Google Scholar 

  34. Tuxworth, R.I., Cheetham, J.L., Machesky, L.M., Spiegelmann, G.B., Weeks, G. & Insall, R.H. Dictyostelium RasG is required for normal motility and cytokinesis, but not growth. J. Cell Biol. 138, 605–614 (1997).

    Article  CAS  Google Scholar 

  35. Jordan, M., Schallhorn, A. & Wurm, F.M. Free in PMC transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601 (1996).

    Article  CAS  Google Scholar 

  36. Loyter, A., Scangos, G.A. & Ruddle, F.H. Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc. Natl. Acad. Sci. USA 79, 422–426 (1982).

    Article  CAS  Google Scholar 

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Acknowledgements

dictyBase (http://www.dictybase.org) is supported by grants from the NIH (GM64426 and HG00022).

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  1. dictyBase, Center for Genetic Medicine, Northwestern University, 676 North Saint Clair Street Suite 1260, Chicago, 60611, Illinois, USA

    Pascale Gaudet, Karen E Pilcher, Petra Fey & Rex L Chisholm

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Correspondence to Rex L Chisholm.

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Gaudet, P., Pilcher, K., Fey, P. et al. Transformation of Dictyostelium discoideum with plasmid DNA. Nat Protoc 2, 1317–1324 (2007). https://doi.org/10.1038/nprot.2007.179

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