Reverse genetics through random mutagenesis in Histoplasma capsulatum
Issue Date:
2009-11-17Metadata
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BioMed CentralCitation:
Brian H. Youseff, Julie A. Dougherty, and Chad A. Rappleye, "Reverse genetics through random mutagenesis in Histoplasma capsulatum," BMC Microbiology 9 (2009), doi:10.1186/1471-2180-9-236, http://www.biomedcentral.com/1471-2180/9/236Abstract:
Background: The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and
systemic disease in humans and other mammals. Progress in understanding the mechanisms
underlying the biology and the pathogenesis of Histoplasma has been hindered by a shortage of
methodologies for mutating a gene of interest.
Results: We describe a reverse genetics process that combines the random mutagenesis of
Agrobacterium-mediated transformation with screening techniques to identify targeted gene
disruptions in a collection of insertion mutants. Isolation of the desired mutant is accomplished by
arraying individual clones from a pool and employing a PCR-addressing method. Application of this
procedure facilitated the isolation of a cbp1 mutant in a North American type 2 strain, a Histoplasma
strain recalcitrant to gene knock-outs through homologous recombination. Optimization of
cryopreservation conditions allows pools of mutants to be banked for later analysis and recovery
of targeted mutants.
Conclusion: This methodology improves our ability to isolate mutants in targeted genes, thereby
facilitating the molecular genetic analysis of Histoplasma biology. The procedures described are
widely applicable to many fungal systems and will be of particular interest to those for which
homologous recombination techniques are inefficient or do not currently exist.
Type:
ArticleISSN:
1471-2180Collections
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