Construction of Double Heterozygous Deletion Strains for Complex Haploinsufficiency-Based Genetic Analysis in Candida...

Complex haploinsufficiency refers to the genetic interaction that occurs in strains with heterozygous mutations at two different loci (a double heterozygous deletion mutant). Double heterozygous deletion mutants can be used to identify gene partners that act within the same pathway or to determine expression-dependent genetic interactions that result in phenotypic changes outside of what would be expected based on the phenotypes of the single heterozygous deletion mutants. The approach outlined here uses a lithium acetate transformation method on a parental "query" strain to introduce a transcription factor deletion DNA construct that is derived from the Homann et al. Candida albicans transcription factor deletion library (Homann et al. PLoS Genet 5(12):e1000783, 2009). We also outline the steps to confirming the genotype of the resulting transformants as well as an example of the use of double heterozygous deletion mutants for complex haploinsufficiency analysis of biofilm formation.