Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition
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Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.
KeywordsGene disruption Transposon Basidiomycetes Virulence factors
Funding was provided by the National Institute of Allergy and Infectious Disease (5RO1AI053721) and the Canadian Institutes of Health Research (CIHR) to JWK. WM276 shotgun sequencing was completed at the Michael Smith Genome Science Centre (GSC) with funding from Genome Canada to JWK. We thank the sequencing group at the GSC for construction of the libraries.
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