Cassettes for PCR-mediated gene tagging in Candida albicans utilizing nourseothricin resistance

Stephen W. Milne, Jill Cheetham, Deborah Lloyd, Stephen J. Aves, Steven Bates

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

In recent years a number of molecular tools have been reported for use in the human fungal pathogen Candida albicans, including PCR-mediated approaches for gene disruption, conditional expression and epitope tagging. Traditionally these methods have utilized auxotrophic markers; however, the availability of auxotrophic markers can be limiting and in some instances their use may also impact on the interpretation of results. As a result, the use of positive selection markers has now become more commonplace. Here we report the development and validation of a set of cassettes for PCR-mediated gene tagging and overexpression studies utilizing the nourseothricin resistance (CaNAT1) positive selection marker. In particular we have produced cassettes containing yeast-enhanced GFP, YFP, CFP, RFP and a combined V5-6xHis epitope tag. The cassettes are engineered for use in PCR-mediated gene tagging strategies where insertion is targeted to the 3' end of the gene of interest. In addition, to facilitate protein functional analysis and genetic suppression studies through the use of overexpression, we have also constructed a promoter replacement cassette containing the ENO1 promoter which is known to be expressed at a high level. These cassettes expand on the range of molecular tools available for working with C. albicans and may also be used in other Candida species that display sensitivity to nourseothricin.

Original languageEnglish
Pages (from-to)833-841
Number of pages9
JournalYeast
Volume28
Issue number12
DOIs
Publication statusPublished - Dec 2011

Fingerprint

Streptothricins
Candida
Candida albicans
Genes
Epitopes
Polymerase Chain Reaction
Genetic Suppression
Functional analysis
Pathogens
Yeast
Yeasts
Availability
Proteins

Keywords

  • Candida albicans
  • Epitope tagging
  • Fluorescent protein
  • Nourseothricin resistance
  • Overexpression
  • Positive selection

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Genetics

Cite this

Cassettes for PCR-mediated gene tagging in Candida albicans utilizing nourseothricin resistance. / Milne, Stephen W.; Cheetham, Jill; Lloyd, Deborah; Aves, Stephen J.; Bates, Steven.

In: Yeast, Vol. 28, No. 12, 12.2011, p. 833-841.

Research output: Contribution to journalArticle

Milne, Stephen W. ; Cheetham, Jill ; Lloyd, Deborah ; Aves, Stephen J. ; Bates, Steven. / Cassettes for PCR-mediated gene tagging in Candida albicans utilizing nourseothricin resistance. In: Yeast. 2011 ; Vol. 28, No. 12. pp. 833-841.
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