Gene Disruption in Aspergillus fumigatus Using a PCR-Based Strategy and In Vivo Recombination in Yeast

Abstract

Aspergillus fumigatus is a ubiquitous, filamentous fungal saprophyte and is the causative agent of the vast majority of aspergillosis in that invasive aspergillosis is the life-threatening form of infection by this fungus. The study of gene function using null mutants in this organism can be achieved through DNA-mediated transformation with an engineered deletion cassette containing about 2 kb of the 5'- and 3'-flanking region of the target gene and a selectable marker. Here, we describe the use of a PCR-based strategy and "in vivo" recombination in Saccharomyces cerevisiae to produce gene deletion cassettes for A. fumigatus, using an auxotrophic marker for gene replacement. This protocol produces highly effective deletion cassettes and permits the rapid disruption of genes identified in the recently available A. fumigatus genome.