title: Reproducible genetic transformation system for two dermatophytes, Microsporum canis and Trichophyton mentagrophytes.
abstract: A reproducible genetic transformation system was developed for two major dermatophytes, Microsporum canis and Trichophyton mentagrophytes. Two circular transformation vectors carrying either the bacterial hygromycin B phosphotransferase gene (hph) or both the hph and green fluorescent protein (eGFP) genes under the control of a promoter sequence from Cochlibolus heterostrophus were introduced independently into the protoplasts by a polyethylene glycol (PEG)-mediated method. Polymerase chain reaction (PCR) showed that the hph gene was integrated randomly into the chromosomal DNA of the transformants through non-homologous recombination. Southern blotting analysis also demonstrated a single or multiple integration of the hph gene into the chromosomal DNA. Fluorescence due to eGFP gene expression was observed in the T. mentagrophytes transformants, and the transformants retained mitotic stability through subculture. This reproducible transformation system provides a method for the genetic manipulation of these pathogens, which will facilitate detailed molecular analysis of dermatophytosis.
authors.affiliation: Teikyo University Institute of Medical Mycology, Hachioji, Tokyo, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamada
authors.forename: Koichi
authors.initials: K
authors.lastname: Makimura
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Uchida
authors.forename: Hideyo
authors.initials: H
authors.lastname: Yamaguchi
mesh_terms: D015139:Blotting, Southern; D004271:DNA, Fungal / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D008864:Microsporum / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic; D014249:Trichophyton / Q000235:genetics*
chemical_list: D004271:DNA, Fungal; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase