Cryptococcus neoformans is an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals and is responsible for more than 1,000,000 infections and 600,000 deaths annually worldwide. Nevertheless, anti-cryptococcal therapeutic options are limited, mainly because of the similarity between fungal and human cellular structures. Owing to advances in genetic and molecular techniques and bioinformatics in the past decade, C. neoformans, belonging to the phylum basidiomycota, is now a major pathogenic fungal model system. In particular, genetic manipulation is the first step in the identification and characterization of the function of genes for understanding the mechanisms underlying the pathogenicity of C. neoformans. This unit describes protocols for constructing target gene deletion mutants using double-joint (DJ) PCR, constitutive overexpression strains using the histone H3 gene promoter, and epitope/fluorescence protein-tagged strains in C. neoformans.
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