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

Wiegand, S. ; Kruse, J. ; Gronemann, S. ; Hammann, Christian (2011)
Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.
In: Genomics, 97 (5)
doi: 10.1016/j.ygeno.2011.02.001
Artikel, Bibliographie

Kurzbeschreibung (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.

Typ des Eintrags: Artikel
Erschienen: 2011
Autor(en): Wiegand, S. ; Kruse, J. ; Gronemann, S. ; Hammann, Christian
Art des Eintrags: Bibliographie
Titel: Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.
Sprache: Englisch
Publikationsjahr: 2011
Titel der Zeitschrift, Zeitung oder Schriftenreihe: Genomics
Jahrgang/Volume einer Zeitschrift: 97
(Heft-)Nummer: 5
DOI: 10.1016/j.ygeno.2011.02.001
Kurzbeschreibung (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.

Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie > Regulatorische RNAs und Ribozyme
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10 Fachbereich Biologie
Hinterlegungsdatum: 23 Feb 2011 14:51
Letzte Änderung: 05 Mär 2013 09:46
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