One-step Targeted Gene Deletion in Candida Albicans Haploids

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetates
  • Candida albicans / genetics*
  • Electroporation
  • Flow Cytometry
  • Gene Deletion*
  • Genetic Engineering / methods*
  • Haploidy*
  • Plasmids / genetics
  • Polymerase Chain Reaction

Substances

  • Acetates
  • lithium acetate