Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus.
Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus.
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DOI：
10.1186/1471-2180-5-1
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发表时间：
2005-01-13
期刊：
影响因子：
4.2
通讯作者：
Askew DS
中科院分区：
文献类型：
--
作者：
Vogt K;Bhabhra R;Rhodes JC;Askew DS
虽然烟曲霉是一种重要的人类真菌病原体，但很少有表达系统可用于研究特定基因对这种机会性霉菌生长和毒力的贡献。基于大肠杆菌四环素抗性操纵子中的原核调控元件的可调节启动子系统已成功地用于操纵几种生物的基因表达，包括小鼠，苍蝇，植物和酵母。然而，该系统尚未适用于曲霉。在这里，我们描述了质粒载体的构建，可以使用简单的共转染方法来调节烟曲霉的基因表达。制备了四环素反激活子（tTA）或逆四环素反激活子（rtTA2s-M2）受A. nidulans gpdA启动子控制的载体。将优势选择带引入每个质粒中，通过在培养基中加入静脉霉素或潮霉素，将基因转移到烟曲霉中进行选择。为了模拟一个受四环素调控的重要基因，将大肠杆菌水霉素耐药基因hph置于质粒载体中七个TetR结合位点（tetO7）的控制下，并与两个反激活质粒中的一个一起共转染到烟曲霉原生质体中。由于hph基因在潮霉素存在下对烟曲霉至关重要，因此对潮霉素的抗性被用作hph报告基因表达的标记。经鉴定，tTA的表达通过激活tetO7-hph报告基因的表达而赋予了潮霉素抗性，并且在培养基中添加多西环素以剂量依赖性的方式抑制了潮霉素抗性。同样，我们也发现了仅在强力霉素存在下rtTA2s-M2表达才产生潮霉素抗性的转化子。调节tetO7-hph报告基因表达所需的强力霉素水平在该生物体的无毒范围内，并且低铁培养基被证明可以减少完成调节所需的强力霉素的量。本报告中描述的载体提供了一套新的选择，以实验方式操纵烟熏革螨特定基因产物的水平
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. 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 (rtTA2s-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 (tetO7) 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 tetO7-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 rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-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. The vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in A. fumigatus
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影响因子：
7.2
作者：
WEINMANN, P;GOSSEN, M;GATZ, C
通讯作者：
GATZ, C
影响因子：
3.1
作者：
Wasylnka, JA;Moore, MM
通讯作者：
Moore, MM
DOI：
10.1073/pnas.130192197
发表时间：
2000-07-05
影响因子：
11.1
作者：
Urlinger, S;Baron, U;Hillen, W
通讯作者：
Hillen, W
影响因子：
5.3
作者：
Stoyan, T;Gloeckner, G;Carbon, J
通讯作者：
Carbon, J
影响因子：
4.9
作者：
Liu, W;May, GS;Kontoyiannis, DP
通讯作者：
Kontoyiannis, DP