New pFA-cassettes for PCR-based gene manipulation in Candida albicans
New pFA-cassettes for PCR-based gene manipulation in Candida albicans
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DOI：
10.1002/jobm.200510133
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发表时间：
2006-01-01
影响因子：
3.1
通讯作者：
Wendland, Jurgen
中科院分区：
文献类型：
--
作者：
Schaub, Yvonne;Dunkler, Alexander;Wendland, Jurgen
最近在白色念珠菌中引入了几个基于聚合酶链式反应的高效基因破坏模块。这些都是基于来自SC5314/CA14的缺陷株的营养缺陷性标记基因。白念珠菌转化的常用方案是基于醋酸锂程序或电穿孔，也用于酿酒酵母。在这里，我们介绍了我们最新的PFA模板库，其中包括来自杜布利尼锦鸡儿的异源标记基因HIS1和来自麦芽锦鸡儿的异源标记基因LEU2(Noble和Johnson 2005)以及显性选择标记ca SAT1(Reuss等人。2004年)。我们还介绍了阿什比亚棉TEF1-启动子作为一种强有力的组成型启动子。利用这些新的元件，产生了一个扩大的PFA标记和PFA标记-启动子模块集合，其中包含17个新模块。此外，还构建了GFP-(GA)6的N端标签和使用6个x-His标签的表位标签模块。这增加了前面的模块，这些模块只支持基因的C-末端GFP标记(Gola等人)。2003年)。我们的实验室目前总共提供了29个PFA模块，加上诊断验证程序的更新，进一步扩大了白念珠菌分子工具箱，并增强了我们在白念珠菌中使用基于PCR的基因改变方法的能力。
Several modules for efficient PCR-based gene disruption have recently been introduced in Candida albicans. These are based on auxotrophic marker genes for deficient strains derived from SC5314/CA14. Commonly used protocols for the transformation C. albicans are based either on the lithium acetate procedure or on electroporation also used for Saccharonzyces cerevisiae. Here we present our updated arsenal of pFA-modules that now include the heterologous marker genes HIS1 from C. dubliniensis and LEU2 from C. maltosa (NOBLE and JOHNSON 2005) and the dominant selection marker ca SAT1 (Reuss et al. 2004). We also introduce the Ashbya gossypii TEF1-promoter as a strong constitutive promoter. With these new elements an enlarged collection of pFA-marker and pFA-marker-promoter modules were generated containing 17 new modules. In addition, N-terminal tagging with GFP-(GA) 6 and epitope-tagging modules using the 6 x-HIS-tag were constructed. This adds to the previous modules that only enabled C-terminal GFP-tagging of genes (GOLA et al. 2003). In total 29 pFA-modules are currently freely available from our lab which - together with an update on the diagnostic verification procedure - further enlarge the C albicans molecular toolbox and enhance our capabilities to use PCR-based gene alteration methods in C. albicans.