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Eukaryotic Cell, February 2005, p. 298-309, Vol. 4, No. 2
1535-9778/05/$08.00+0     doi:10.1128/EC.4.2.298-309.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen Candida albicans

Suzanne M. Noble* and Alexander D. Johnson

Department of Microbiology and Immunology, University of California-San Francisco, San Francisco, California

Received 16 March 2004/ Accepted 19 November 2004

Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2{Delta}/leu2{Delta}, his1{Delta}/his1{Delta}; arg4{Delta}/arg4{Delta}, his1{Delta}/his1{Delta}; and arg4{Delta}/arg4{Delta}, leu2{Delta}/leu2{Delta}, his1{Delta}/his1{Delta}) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.

* Corresponding author. Mailing address: Dept. of Microbiology & Immunology, University of California-San Francisco, 600 16th St., Box 2200, San Francisco, CA 94143-2200. Phone: (415) 502-0859. Fax: (415) 502-4315. E-mail: snoble{at}itsa.ucsf.edu.

Eukaryotic Cell, February 2005, p. 298-309, Vol. 4, No. 2
1535-9778/05/$08.00+0     doi:10.1128/EC.4.2.298-309.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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