English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Background  Although  Aspergillus fumigatus  is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the  E. coli  tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for  Aspergillus spp .    Results  Here we describe the construction of plasmid vectors that can be used to regulate gene expression in  A. fumigatus  using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2 s -M2) are controlled by the  A. nidulans gpdA  promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into  A. fumigatus  by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the  E. coli  hygromycin resistance gene,  hph , was placed under the control of seven copies of the TetR binding site ( tetO  7 ) in a plasmid vector and co-transfected into  A. fumigatus  protoplasts together with one of the two transactivator plasmids. Since the  hph  gene is essential to  A. fumigatus  in the presence of hygromycin, resistance to hygromycin was used as a marker of  hph  reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the  tetO  7 - hph  reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2 s -M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the  tetO  7 - hph  reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation.    Conclusions  The vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in  A. fumigatus

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus.