title: Genome-wide synthetic genetic screening by transposon mutagenesis in Candida albicans.
abstract: Transposon-based mutagenesis is an effective method for genetic screening on a genome-wide scale, with particular applicability in organisms possessing compact genomes where transforming DNA tends to integrate by homologous recombination. Methods for transposon mutagenesis have been applied with great success in the budding yeast Saccharomyces cerevisiae and in the related pathogenic yeast Candida albicans. In C. albicans, we have implemented transposon mutagenesis to generate heterozygous mutations for the analysis of complex haploinsufficiency, a type of synthetic genetic interaction wherein a pair of non-complementing heterozygous mutations results in a stronger phenotype then either individual mutation in isolation. Genes exhibiting complex haploinsufficiency typically function within a regulatory pathway, in parallel pathways, or in parallel branches within a single pathway. Here, we present protocols to implement transposon mutagenesis for complex haploinsufficiency screening in C. albicans, indicating methods for transposon construction, mutagenesis, phenotypic screening, and identification of insertion sites in strains of interest. In total, the approach is a useful means to implement large-scale synthetic genetic screening in the diploid C. albicans.
authors: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109-1048, USA.
authors: Brooke N
authors: BN
authors: Horton
authors: Anuj
authors: A
authors: Kumar
mesh_terms: D000483:Alleles; D002176:Candida albicans / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D015723:Gene Library; D005813:Genes, Synthetic; D005820:Genetic Testing / Q000379:methods*; D016681:Genome, Fungal; D057895:Haploinsufficiency / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010641:Phenotype; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements
references: Eukaryot Cell. 2003 Oct;2(5):1053-60
references: 14555488
references: Cell. 1997 Sep 5;90(5):939-49
references: 9298905
references: Science. 2001 Dec 14;294(5550):2364-8
references: 11743205
references: Eukaryot Cell. 2012 Jun;11(6):708-17
references: 22544903
references: PLoS Genet. 2009 Dec;5(12):e1000783
references: 20041210
references: Eukaryot Cell. 2008 Aug;7(8):1289-98
references: 18310357
references: Nucleic Acids Res. 2000 Mar 1;28(5):1067-77
references: 10666445
references: Nucleic Acids Res. 2006 Apr 28;34(8):e57
references: 16648358
references: EMBO J. 1997 Nov 17;16(22):6823-34
references: 9362496
references: Genome Res. 2004 Oct;14(10A):1975-86
references: 15466296
references: Nat Genet. 2010 Jul;42(7):590-8
references: 20543849
references: Genetics. 1997 Mar;145(3):573-85
references: 9055068
references: Methods Enzymol. 2002;350:230-47
references: 12073315
references: Genes Dev. 1994 May 1;8(9):1087-105
references: 7926789
references: Genetics. 1988 Jun;119(2):249-60
references: 3294100
references: EMBO J. 2003 Jun 2;22(11):2668-78
references: 12773383
references: Mol Biol Cell. 2008 Dec;19(12):5456-77
references: 18843050
references: Genes Dev. 2007 Jan 15;21(2):148-59
references: 17167106
references: Methods Mol Biol. 2006;313:171-92
references: 16118434
references: PLoS Pathog. 2010 Feb 05;6(2):e1000752
references: 20140194
references: Science. 1999 Aug 6;285(5429):901-6
references: 10436161
references: Nature. 1999 Nov 25;402(6760):413-8
references: 10586881
references: J Bacteriol. 2002 Apr;184(7):2058-61
references: 11889116
references: Mol Gen Genet. 1981;182(3):516-9
references: 6795427
references: Genetics. 2002 Dec;162(4):1573-81
references: 12524333
references: PLoS Genet. 2011 Apr;7(4):e1002058
references: 22103005