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Cassettes for PCR‐mediated gene tagging in Candida albicans utilizing nourseothricin resistance
Author(s) -
Milne Stephen W.,
Cheetham Jill,
Lloyd Deborah,
Aves Stephen,
Bates Steven
Publication year - 2011
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1910
Subject(s) - biology , candida albicans , gene , genetics , resistance (ecology) , computational biology , microbiology and biotechnology , ecology
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. Copyright © 2011 John Wiley & Sons, Ltd.