The adaptation of the Cre-loxP gene disruption system for use in Candida albicans
At the start of this project, the systems available for gene disruption in C. albicans were laborious and inefficient. Both alleles of a target locus need to be inactivated to construct a homozygous null mutant in this diploid fungus. This often requires the recycling of transformation markers. However, in some systems it is not possible to recycle the markers used in the disruption. This limited the creation of multiple knockout strains and the deletion of gene families. This thesis describes the successful adaptation of the Cre-loxP recombination system as tool for the precise, efficient and controlled disruption of genes in C. albicans. LoxP-MARKER-loxP cassettes containing URA3, HIS1, and ARG4 genes were constructed for gene disruption. Also, ano arg4::MET3p-cre-URA3 cassette was generated for loxP-flanked marker resolution. This cassette contains a functional, codon-optimised cre gene. Finally plasmids containing the URA3, HIS1, and ARG4 genes were constructed for the restoration of prototrophy and the re-integration of target genes into C. albicans null mutants. The usefulness of the Cre-loxP system was demonstrated by the generation of homozygous ade2D and met15D null mutants, using loxP flanked ARG4 and HIS1 based disruption cassettes. A third cassette containing URA3 and a MET3p-cre recombinase fusion was integrated within the loxP-ARG4-loxP cassette. This facilitated the resolution of the MET3p-cre fusion and the loxP-flanked disruption cassettes from the C. albicans genome by Cre-mediated site-specific recombination between the flanking loxP elements. The system resolved the cassettes with near 100% efficiency. This allows further manipulations to be performed on the resultant null mutant. Therefore this new Cre-loxP system could potentially be used for the deletion of gene families in C. albicans.