A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans

Wei Chung Lai, Hsiao Fang Sunny Sun, Pei Hsuan Lin, Ho Lin, Jia Ching Shieh

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Candida albicans is an important human fungal pathogen but its study has been hampered for being a natural diploid that lacks a complete sexual cycle. Gene knock-out and essential gene repression are used to study gene function in C. albicans. To effectively study essential genes in wild-type C. albicans, we took advantage of the compatible effects of the antibiotics hygromycin B and nourseothricin, the recyclable CaSAT1-flipper and the tetracycline-repressible (Tet-off) system. To allow deleting two alleles simultaneously, we created a cassette with a C. albicans HygB resistance gene (CaHygB) flanked with the FLP recombinase target sites that can be operated alongside the CaSAT1-flipper. Additionally, to enable conditionally switching off essential genes, we created a CaHygB-based Tet-off cassette that consisted of the CaTDH3 promoter, which is used for the constitutive expression of the tetracycline-regulated transactivator and a tetracycline response operator. To validate the new systems, all strains were constructed based on the wild-type strain and selected by the two dominant selectable markers, CaHygB and CaSAT1. The C. albicans general transcriptional activator CaGCN4 and its negative regulator CaPCL5 genes were targeted for gene deletion, and the essential cyclin-dependent kinase CaPHO85 gene was placed under the Tet-off system. Cagcn4, Capcl5, the conditional Tet-off CaPHO85 mutants, and mutants bearing two out of the three mutations were generated. By subjecting the mutants to various stress conditions, the functional relationship of the genes was revealed. This new system can efficiently delete genes and conditionally switch off essential genes in wild-type C. albicans to assess functional interaction between genes.

Original languageEnglish (US)
Pages (from-to)213-235
Number of pages23
JournalCurrent Genetics
Volume62
Issue number1
DOIs
StatePublished - Feb 1 2016

Fingerprint

Candida albicans
Essential Genes
Genes
Tetracycline
Streptothricins
Hygromycin B
Switch Genes
Gene Knockout Techniques
Trans-Activators
Cyclin-Dependent Kinases
Gene Deletion
Regulator Genes
Diploidy
Alleles
Anti-Bacterial Agents
Mutation

Keywords

  • CaGCN4
  • CaHygB
  • Candida albicans
  • CaPCL5
  • CaPHO85
  • CaSAT1-flipper
  • Tet-off system

ASJC Scopus subject areas

  • Genetics

Cite this

A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans. / Lai, Wei Chung; Sun, Hsiao Fang Sunny; Lin, Pei Hsuan; Lin, Ho; Shieh, Jia Ching.

In: Current Genetics, Vol. 62, No. 1, 01.02.2016, p. 213-235.

Research output: Contribution to journalArticle

Lai, Wei Chung ; Sun, Hsiao Fang Sunny ; Lin, Pei Hsuan ; Lin, Ho ; Shieh, Jia Ching. / A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans. In: Current Genetics. 2016 ; Vol. 62, No. 1. pp. 213-235.
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