A Nonlethal Murine Cutaneous Model of Invasive Aspergillosis
Cutaneous models allow researchers to dynamically monitor infection by visually determining changes in skin lesion dimensions over time. We present a nonlethal cutaneous model of invasive aspergillosis (IA) in nude BALB/c mice and describe its use to dete
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1. Introduction Candida albicans is a diploid organism without a known haploid phase. To create mutants lacking a specific gene, both alleles of the gene have to be sequentially inactivated. This is usually performed by transforming C. albicans cells with linear DNA fragments containing a selection marker that is flanked by the upstream and downstream sequences of the target gene. Homologous recombination then results in replacement of one of the wild-type alleles in 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_1, © Springer Science+Business Media, LLC 2012
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C. Sasse and J. Morschhäuser
the genome by the selection marker. Gene deletions are often performed in auxotrophic host strains with defects in certain biosynthetic pathways (e.g., for arginine, histidine, and uracil) that can be complemented using the corresponding intact genes (e.g., ARG4, HIS1, or URA3) as selection markers. Homozygous mutants are obtained either by using different markers for the inactivation of the two alleles in a host strain with multiple auxotrophies or by repeated use of a counterselectable, recyclable marker (e.g., URA3) for sequential deletion of both alleles of the target gene (1). These methods are relatively straightforward, but they also have drawbacks. First, their use is limited to laboratory strains with appropriate auxotrophies. Therefore, they cannot be applied to study the role of specific genes in clinical isolates, which are usually prototrophic. Yet, sometimes, the contribution of a gene to a certain phenotype may not be revealed in laboratory strains. For example, the multidrug efflux pump MDR1 is not significantly expressed in most strains under standard growth conditions, but it is constitutively overexpressed in drug-resistant clinical isolates that have acquired gain-of-function mutations in its regulator, the transcription factor MRR1. While the inactivation of MDR1 or MRR1 in a laboratory strain had no effect on drug susceptibility, the role of these genes in drug resistance could be clearly demonstrated by deleting them in MDR1 overexpressing clinical isolates (2–5). Second, the introduction of biosynthetic markers into an auxotrophic host strain that does not contain these genes results in mutants that cannot be directly compared with their parental strain, because they differ not only by the absence of the target gene but also by the presence of the selection markers. Therefore, this strategy requires the inclusion of other control strains that contain the same markers. Despite this, mutants and wild-type control strains still usually differ by the genomic location of the markers (the target locus in the mutants and the native locus or another integration site in the control strain), which can affect the expression level of the marker and the phenotype of the cells (6–8). When a counterselectable, recyclable marker like URA3 is used for sequential gene disruptions, the resulting mut
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