Notably, Yamada and colleagues used the system for both random integration of T-

(transferred) DNA and targeted insertion, for example disruption of the areA/nit-2 gene. As another alternative transformation technique, electroporation of germinated conidia was applied in T. rubrum, allowing the random integration of hph and eGFP (Dobrowolska & Staczek, 2009). Although not many comparative data on Thiazovivin clinical trial transformation efficiency are available – some species have not even been addressed at all – different dermatophyte species appear to be more or less amenable to DNA uptake and/or stable integration. Therefore, transformation protocols established for a selected species are not necessarily transferable to another, but require precise modifications. From our own work, we know for example that our standard PEG-protocol for the efficient transformation of A. benhamiae was not directly applicable for T. rubrum or M. canis.

The reasons for this observation are likely multifactorial, http://www.selleckchem.com/products/ABT-263.html including differential protoplast stability, cell wall composition, microconidia production, etc. Filamentous fungi are known to only poorly support site-directed insertion of linear DNA cassettes in the genome by homologous recombination, in contrast to yeasts such as Saccharomyces cerevisiae or the opportunistic pathogen C. albicans. Therefore, in filamentous fungi, identification of transformants with a desired genetic alteration has proven laborious in many cases. In order to circumvent this obstacle, parental strains were generated in diverse species that lack the nonhomologous end joining (NHEJ) recombination pathway, for example in N. crassa (Ninomiya et al., 2004), Aspergillus

spp. (da Silva Ferreira et al., 2006; Krappmann et al., 2006; Nayak et al., 2006), and since recently, also in T. mentagrophytes (Yamada et al., 2009a) and A. benhamiae Cobimetinib molecular weight (Grumbt et al., 2011) (Table 1). Mutants deficient in NHEJ processes allow a strongly increased frequency of targeted insertions; however, an altered risk of unforeseen genetic variations cannot be excluded. In dermatophyte species, only a small number of genes have so far been analysed by targeted inactivation, for example pacC and MDR2 in T. rubrum (Fachin et al., 2006; Ferreira-Nozawa et al., 2006), Ku80, areA and Trim4 in T. mentagrophytes (Yamada et al., 2009a, b), areA in M. canis (Yamada et al., 2006) and Ku70 and AcuE in A. benhamiae (Grumbt et al., 2011). Interestingly, A. benhamiae has been shown in our work to allow efficient targeted gene deletion not only in a ku70 mutant background but also in the wild-type strain. This has been demonstrated by the construction of mutants in malate synthase AcuE, KU70 and other candidates (Grumbt et al., 2011; M. Grumbt and P. Staib, unpublished data). The use of two different dominant selection markers, hph and neo, even allowed for the first time the site-directed complementation of knockout mutant strains. Because the deletion of KU70 had no adverse effect on the virulence of A.