title: Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.
abstract: The amoeba Dictyostelium discoideum is a well-established model organism for studying numerous aspects of cellular and developmental functions. Its rather small (~34Mb) chromosomal genome and the high efficiency of gene disruption by homologous recombination have enabled researchers to dissect various specific gene functions. We describe here the use of one-step cloning for the fast and efficient generation of deletion vectors that are produced in a one-step reaction by inserting two PCR products into an organism-specific, generic acceptor system. This worked efficiently for all 16 tested constructs directed against genes in the amoeba Dictyostelium discoideum. Saving cost and time, the used protocol represents a significant advancement in the generation of such plasmids compared to the conventionally applied restriction enzyme/ligation approach. Using appropriate selection markers, similar systems could also be useful in other organisms, where genes can be knocked out by homologous recombination.
authors.affiliation: Department for Microbiology and Genetics, Technische Universität Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Wiegand
authors.forename: Janis
authors.initials: J
authors.lastname: Kruse
authors.forename: Sina
authors.initials: S
authors.lastname: Gronemann
authors.forename: Christian
authors.initials: C
authors.lastname: Hammann
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004023:Dictyostelium / Q000235:genetics*; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods