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Genomics. 2011 May;97(5):321-5. doi: 10.1016/j.ygeno.2011.02.001. Epub 2011 Feb 19.

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

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Department for Microbiology and Genetics, Technische Universität Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.


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.

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