One-step targeted gene deletion in Candida albicans haploids.
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.
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.
Guisheng
G
Zeng
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.
Yan-Ming
YM
Wang
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.
Fong Yee
FY
Chan
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.
Yue
Y
Wang
D000085:Acetates; D002176:Candida albicans / Q000235:genetics*; D018274:Electroporation; D005434:Flow Cytometry; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D006238:Haploidy; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
D000085:Acetates; C488804:lithium acetate