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  • Title: Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.
    Author: Wiegand S, Kruse J, Gronemann S, Hammann C.
    Journal: Genomics; 2011 May; 97(5):321-5. PubMed ID: 21316445.
    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.
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