Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.

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.

• Publication year

  2011

• Venue

  Genomics

• Publication date

  2011-05-01

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1016/j.ygeno.2011.02.001PMID 21316445
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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