Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 390, Issue 3, Pages 983-988Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2009.10.089
Keywords
Biolistic transformation; Cryptococcus neoformans; Double-joint PCR; Fungal pathogen; Gene disruption; NAT; Overlap-PCR; Recombination; Split marker
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Funding
- Korea government (MEST) [R01-2008-000-11426-0, R11-2008-062-02001-0]
- National Research Foundation of Korea [R11-2008-062-02001-0, R01-2008-000-11426-0, 2008-0061963, 2008-0058681] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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Targeted gene disruption via biolistic transformation and homologous recombination is a method widely used to identify and investigate the function of genes in Cryptococcus neoformans that causes fatal fungal meningitis if not timely treated. Currently, most laboratories employ the overlap-PCR method to generate a gene-disruption cassette with dominant selectable markers, such as nourseothricin acetyltransferase (NAT). However, the conventional overlap-PCR method is often found to be inefficient because of the presence of multiple templates and of the long length of the final overlap-PCR products. In this report, we suggested an efficient gene-disruption method for C. neoformans, termed a double-joint PCR with NAT-split markers. Here we demonstrated that the gene-disruption cassette generated using double-joint PCR with NAT-split markets can be used Successfully for targeted C neoformans gene disruption with the advantages of providing a more convenient construction of gene-disruption cassettes and high targeted-integration frequency. (C) 2009 Elsevier Inc. All rights reserved.
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