One-step targeted gene deletion in Candida albicans haploids

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One-step targeted gene deletion in Candida albicans haploids
Title:
One-step targeted gene deletion in Candida albicans haploids
Journal Title:
Nature Protocols
Keywords:
Publication Date:
30 January 2014
Citation:
Nature Protocols 9, 464–473 (2014)
Abstract:
The recent discovery of haploids in Candida albicans and the construction of tool strains carrying multiple auxotrophic markers have enabled, for the first time, performing one-step gene deletions in this fungal human pathogen. This breakthrough promises to greatly facilitate the molecular and genetic study of C. albicans biology and pathogenicity. However, the construction of gene-deletion mutants in C. albicans haploids involves many technical difficulties, particularly low transformation efficiency and autodiploidization. Here we describe a highly effective protocol for designing and performing one-step gene deletion in C. albicans haploids, which takes ∼11 d to complete (not including plasmid construction, which may take ∼2 weeks). A gene deletion cassette is constructed on a plasmid and subsequently released for transformation by lithium acetate incubation or electroporation. Desired gene-deletion mutants are identified and their ploidy is assessed simultaneously by colony PCR before final confirmation by flow cytometry.
License type:
PublisherCopyrights
Funding Info:
Description:
ISSN:
1754-2189
1750-2799
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