Mini-blaster-Mediated Targeted Gene Disruption and Marker Complementation in Candida albicans
Several gene disruption strategies have been described in Candida albicans to create homozygous mutants. We describe here a recyclable mini-blaster cassette containing C. albicans URA3 gene and 200-bp flanking repeats that is useful for disruption of C. a
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1. Introduction Candida albicans is a major fungal systemic pathogen in humans. The ability of this fungus to cause lethal infections has fueled the need to study its gene function to better understand aspects such as host-pathogen interactions and virulence. The genetic architecture of this pathogen poses two significant obstacles (1). First, C. albicans is a diploid so both alleles of a gene must be altered. Second, it is an asexual organism, so gene disruption and complementation depends upon successive manipulations of a single strain. Loss-of-function or null mutations provide a simple avenue toward interpretation of gene function. In this chapter, we discuss a detailed protocol to introduce a null mutation in a C. albicans gene of interest. We describe use of the mini-blaster strategy to delete both alleles of a gene by alternate transformation and recombination using a recyclable cassette containing the C. albicans URA3 marker (2). This strategy is based on the original Ura-blaster design of Alani and Kleckner (3) that was modified for C. albicans
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_2, © Springer Science+Business Media, LLC 2012
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S. Ganguly and A.P. Mitchell Mini Blaster Cassette in pDDB57 plasmid
URA3
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3.0 kb 2.0 kb 1.5 kb
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3
4
5
Mini Blaster Product
1.0 kb 0.5 kb
Loop Out Product
Fig. 1. PCR amplification of the mini-blaster cassette from the pDDB57 plasmid. Gray boxes in the diagram indicate the 200 bp repeats flanking the URA3 ORF. In the gel picture, lanes 1–5 show the ~2,000-bp long cassette and a ~750-bp long loop-out product.
by Fonzi and Irwin (4). The mini-blaster, or “Ura-blaster,” has shorter direct repeats than the Ura-blaster, thus facilitating PCR amplification. Specifically, the mini-blaster cassette carries the URA3-dpl200 marker (Fig. 1), which constitutes the URA3 gene and 200-bp flanking repeats. The flanking repeats permit homologous excision and reutilization of the URA3 marker. The cassette is amplified and targeted using primers bearing 100 bp of homology to the gene locus. These same primers may be used for a more conventional dual-marker gene disruption procedure (5). Transformation with the PCR product confers a Ura + phenotype, creating a heterozygous mutant with one allele of the targeted gene replaced by the cassette. Subsequent growth of the Ura + heterozygote under nonselective conditions, followed by selection on 5-FOA (5-fluoroorotic acid) plates, yields a Ura− heterozygous strain. 5-FOA is used for identification and selection of cells that have undergone spontaneous loss of URA3 to become Ura−. Cells that have retained URA3 can synthesize the enzyme orotidine-5¢-phosphate decarboxylase, which converts 5-FOA into a toxic compound and therefore renders Ura + cells unable to grow on plates containing 5-FOA. Transformation and 5-FOA selection are repeated with the Ura− heterozygote to delete the second allele of the gene,
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