title: Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei.
abstract: Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway™ system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.
authors.affiliation: Department of Genetics, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Hayley E
authors.initials: HE
authors.lastname: Bugeja
authors.forename: Kylie J
authors.initials: KJ
authors.lastname: Boyce
authors.forename: Harshini
authors.initials: H
authors.lastname: Weerasinghe
authors.forename: Sally
authors.initials: S
authors.lastname: Beard
authors.forename: Anne
authors.initials: A
authors.lastname: Jeziorowski
authors.forename: Shivani
authors.initials: S
authors.lastname: Pasricha
authors.forename: Michael
authors.initials: M
authors.lastname: Payne
authors.forename: Lena
authors.initials: L
authors.lastname: Schreider
authors.forename: Alex
authors.initials: A
authors.lastname: Andrianopoulos
mesh_terms: D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D059765:Homologous Recombination; D006801:Humans; D009181:Mycoses / Q000382:microbiology*; D010407:Penicillium / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; C000607893:LIG4 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP