New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region.
The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.
Gola
Susanne
S
Department of Microbiology, Friedrich-Schiller-University, Jena, Germany.
Martin
Ronny
R
Walther
Andrea
A
Dünkler
Alexander
A
Wendland
Jürgen
J
D002176:Candida albicans / Q000235:genetics*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D014170:Transformation, Genetic / Q000502:physiology
D004271:DNA, Fungal; D005656:Fungal Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins