Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis.
Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis.
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DOI：
10.1002/cpmc.31
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发表时间：
2017-05-16
影响因子：
--
通讯作者：
Fields, Kenneth A
中科院分区：
文献类型：
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作者：
Mueller, Konrad E;Wolf, Katerina;Fields, Kenneth A
基因失活是建立蛋白质功能的正向和反向遗传方法所必需的。诸如插入诱变或化学诱变等技术已经被开发出来，分别通过靶向诱变或随机诱变来诱变衣原体。这两种方法都需要对衣原体进行转化以引入插入元件或补体突变体。我们最近开发了一种靶向诱变策略，荧光报告等位基因交换诱变（FRAEM），以删除沙眼衣原体L2基因。这种方法克服了基因操纵胞内细菌的几个障碍。也许最重要的是，FRAEM采用荧光报告来指示成功的转化和随后的重组事件。三种方案提供了详细的方法来构建基因特异性自杀载体，转化沙眼衣原体L2以选择重组体，并通过限制稀释分离克隆群体。总的来说，这些方案将允许研究人员设计携带所需基因完全缺失的沙眼衣原体L2株。©2017 by John Wiley & Sons, Inc。
Gene inactivation is essential for forward and reverse genetic approaches to establish protein function. Techniques such as insertion or chemical mutagenesis have been developed to mutagenize chlamydiae via targeted or random mutagenesis, respectively. Both of these approaches require transformation of chlamydiae to either introduce insertion elements or complement mutants. We have recently developed a targeted mutagenesis strategy, fluorescence-reported allelic exchange mutagenesis (FRAEM), to delete Chlamydia trachomatis L2 genes. This approach overcomes several barriers for genetically manipulating intracellular bacteria. Perhaps most significantly, FRAEM employs fluorescence reporting to indicate successful transformation and subsequent recombination events. Three protocols are provided that detail methods to construct gene-specific suicide vectors, transform C. trachomatis L2 to select for recombinants, and isolate clonal populations via limiting dilution. In aggregate, these protocols will allow investigators to engineer C. trachomatis L2 strains carrying complete deletions of desired gene(s). © 2017 by John Wiley & Sons, Inc.