Targeted Gene Disruption in Cryptococcus neoformans Using Double-Joint PCR with Split Dominant Selectable Markers
Cryptococcus neoformans causes fatal meningoencephalitis if not timely treated. Targeted gene disruption for functional analysis of a gene involves overlap PCR for the production of gene disruption cassettes carrying dominant selectable markers, followed
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1. Introduction Characterizing the function of a gene usually requires targeted gene disruption by homologous recombination. In the basidiomycetous human fungal pathogen Cryptococcus neoformans, the overlap PCR/biolistic transformation method is well established for targeted gene disruption (1–3). For the selection of transformants, dominant selectable markers, including the nourseothricin acetyltransferase gene (NAT), the neomycin resistance gene (NEO), or the hygromycin resistance gene (HYG) (4, 5), are commonly used. Despite its success for gene disruption in C. neoformans, the overlap PCR method is often inefficient due to the long length of the PCR
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_5, © Springer Science+Business Media, LLC 2012
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product and the presence of multiple templates. In addition, it is rather time consuming. In contrast, double-joint PCR (DJ-PCR) with split dominant selectable markers has proved to be a more convenient and efficient means for the construction of genedisruption cassettes, with higher targeted integration frequency than the conventional overlap PCR-mediated gene disruption (6). DJ-PCR with split dominant selectable markers avoids most of problems imposed by conventional overlap PCR for the following reasons. First, only two template PCR fragments (approx. 0.7–1 kb of the 5¢- or 3¢-flanking regions of the target gene plus 1 kb of the split marker) are used for DJ-PCR. These, truncated disruption cassettes are more efficient to generate because the shorter PCR fragments require less amplification time (~2 kb in size; 2:30 min) than the conventional overlap PCR (~4 kb in size; 4:30 min). Second, DJ-PCR with split dominant selectable markers does not require any gel-extraction steps during the process and, therefore, saves additional time and labor in the preparation of gene disruption cassettes. Furthermore, targeted integration frequency appears to be higher in the NAT-split marker transformation than that of conventional overlap PCR transformation. Truncated NAT-split markers are less likely to be ectopically integrated by generating the intact NAT marker in the nonnative genomic locus, although integration frequency may be influenced by this locus. The major weakness in the DJ-PCR with NAT-split marker method is that NATr colonies appear more slowly after biolistic cotransformation (3–4 days) than the conventional overlap PCR (2–3 days). It is possible that NAT-split markers require more time for additional recombination inside the cell. Regardless of these concerns, DJ-PCR with NAT-split marker transformation is the method of choice for large-scale gene disruption in C. neoformans. In conclusion, DJ-PCR with NAT-split markers-mediated transformation is a very efficient and easy-to-perform gene disruption method in C. neoformans. 1.1. Safe Handling of C. neoformans
Cryptococcus neoformans is classified as Biosafety Level 2 (BL2)
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