Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei

Talaromyces marneffeilaccase modifies THP-1 macrophage responses

“…G681 is a derivative of the type strain FRR2161. 54 All transformants and G681 strain were generated from G526 (Dpku pyrG ¡ ) stain which kuA is deleted to inhibit non-homologous DNA end joining repair. All transformants were grown at 28 C on Aspergillus nidulans synthetic medium (ANM).…”

Thermally Dimorphic Human Fungal Pathogens—Polyphyletic Pathogens with a Convergent Pathogenicity Trait

“…These tools have been used to probe the molecular mechanisms that control the dimorphic switch and allow the fungus to survive within the host. These include (1) a very high frequency DNA-mediated transformation procedure using PEG-based protoplast fusions that results in integration of exogenous DNA (Borneman et al 2001), (2) dominant selectable markers with matching recipient strains for transformation , (3) enhanced strains in which DNA integration is strictly by homologous recombination using mutants in the nonhomologous endjoining (NHEJ) system , (4) strains expressing fluorescent proteins for livecell tracking , (5) antisense RNA knockdown systems (Canovas et al 2011), (6) gateway systems for rapid construct generation Bugeja et al 2012), (7) promoters for controlled expression of genes (Borneman et al 2000;Boyce et al 2001), (8) genomic and transcriptomic technologies (Pasricha et al 2013), and (9) cell culture, zebrafish, and mouse-based systems for virulence testing (Boyce and Andrianopoulos 2007;Ellett et al 2011;Henk et al 2012). A procedure for DNAmediated transformation using the Agrobacterium transfer-DNA system has also been described (Kummasook et al 2010).…”

Strategies for the molecular genetic manipulation and visualization of the human fungal pathogen Penicillium marneffei

“…Selectable markers available for the generation of deletion constructs using a Gateway TM cloning system A pipeline for the cloning and functional characterization of genes in P. marneffei utilizing a Gateway TM cloning system to facilitate the rapid generation of gene deletion constructs has been developed (Bugeja et al, 2012). This approach uses a PCR and recombination based system where the flanking regions of genes to be deleted are amplified by inverse PCR to incorporate attB recognition sequences, which facilitates integration of a selectable marker by in vitro recombination with corresponding attP sequences.…”

“…Combined with the dominant selectable marker of bleomycin/phleomycin resistance and the ability to recycle the pyrG marker (Borneman et al 2001), complex genetically modified strains can be created for analysis. Exogenous DNA introduced during transformation preferentially integrates into the genome of P. marneffei by non-homologous integration, however, strains defective in the non-homologous end-joining machinery have recently been developed that result in highly efficient homologous integration (Table 1) (Bugeja et al, 2012). This study describes the development of additional auxotrophic and dominant selectable markers to broaden the options for selection of transformants containing introduced DNA in the type strain of P. marneffei or into clinical isolates (Table 2).…”

“…This study describes the development of additional auxotrophic and dominant selectable markers to broaden the options for selection of transformants containing introduced DNA in the type strain of P. marneffei or into clinical isolates (Table 2). In addition, a number of constructs have been developed for targeted integration at specific loci and for the rapid generation of gene deletion constructs using the previously described Gateway TM cloning system (Bugeja et al, 2012). Lastly, tools for the microscopic visualization of P. marneffei mutants generated by these techniques are described.…”