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

Wiegand, S. and Kruse, J. and Gronemann, S. and Hammann, Christian (2011):
Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.
In: Genomics, pp. 321-325, 97, (5), ISSN 1089-8646, [Article]

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.

Item Type: Article
Erschienen: 2011
Creators: Wiegand, S. and Kruse, J. and Gronemann, S. and Hammann, Christian
Title: Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.
Language: English
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.

Journal or Publication Title: Genomics
Volume: 97
Number: 5
Divisions: 10 Department of Biology > Ribogenetics
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10 Department of Biology
Date Deposited: 23 Feb 2011 14:51
Identification Number: doi:10.1016/j.ygeno.2011.02.001
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