Journal
MICROBIOLOGY-SGM
Volume 148, Issue -, Pages 2607-2615Publisher
MICROBIOLOGY SOC
DOI: 10.1099/00221287-148-8-2607
Keywords
gene disruption; PCR overlap; Cryptococcus neoformans
Categories
Funding
- NIAID NIH HHS [AI39115S1, R01 AI50115, P01 AI44975, R01 AI39115, R01 AI50438, U01 AI47087, U01 AI48594] Funding Source: Medline
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Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.
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