Journal
EUKARYOTIC CELL
Volume 4, Issue 2, Pages 298-309Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/EC.4.2.298-309.2005
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Funding
- NIAID NIH HHS [R01 AI49187, R01 AI049187] Funding Source: Medline
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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 (leu2Delta/leu2Delta, his1Delta/his1Delta; arg4Delta/arg4Delta, his1Delta/his1Delta; and arg4Delta/arg4Delta, leu2Delta/leu2Delta, his1Delta/his1Delta) that exhibit wild-type or nearly wildtype 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.
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