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Nat Protoc. 2014 Feb;9(2):464-73. doi: 10.1038/nprot.2014.029. Epub 2014 Jan 30.

One-step targeted gene deletion in Candida albicans haploids.

Author information

1
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.
2
1] Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore. [2] Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

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.

PMID:
24481273
DOI:
10.1038/nprot.2014.029
[Indexed for MEDLINE]

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