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dc.creatorYouseff, Brian H.
dc.creatorDougherty, Julie A.
dc.creatorRappleye, Chad A.
dc.date.accessioned2010-11-05T20:49:09Z
dc.date.available2010-11-05T20:49:09Z
dc.date.issued2009-11-17
dc.identifier.citationBrian 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/1471-2180/9/236en_US
dc.identifier.issn1471-2180
dc.identifier.urihttp://hdl.handle.net/1811/47273
dc.description.abstractBackground: 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.en_US
dc.language.isoen_USen_US
dc.publisherBioMed Centralen_US
dc.titleReverse genetics through random mutagenesis in Histoplasma capsulatumen_US
dc.typeArticleen_US
dc.identifier.doi10.1186/1471-2180-9-236
dc.identifier.osuauthorrappleye.1
dc.identifier.osuauthoryouseff.1
dc.identifier.osuauthordougherty.138
dc.rights.ccAttribution 3.0 Unporteden_US
dc.rights.ccurihttp://creativecommons.org/licenses/by/3.0/en_US


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