Published online 26 October 2004
Nucleic Acids Research, Vol. 32 No. 19 © Oxford University Press 2004; all rights reserved
A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system
A. Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität München, D-80336 München, Germany, 1 Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892-8028, USA and 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
* To whom correspondence should be addressed. Tel: +1 301 496 3016; Fax: +1 301 496 5239; Email: ark1{at}helix.nih.gov
Present address: Lisa Kreppel, MedImmune, Inc., Gaithersburg, MD 20878, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received July 21, 2004; Revised and Accepted September 19, 2004
Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small (
34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.
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