Keb Gabrielle, Fields Kenneth A
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky.
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky;
J Vis Exp. 2020 Jan 30(155). doi: 10.3791/60848.
Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.
沙眼衣原体是一种专性胞内病原体，在历史上一直难以进行基因操作。由于缺乏遗传工具，在阐明沙眼衣原体用于创建和维持特殊胞内生态位的机制方面，确切的进展有限。幸运的是，最近基因操作技术有了一些新进展。其中包括荧光报告等位基因交换诱变（FRAEM）的发展。这种方法允许进行靶向基因缺失，并插入一个编码抗生素抗性和绿色荧光蛋白（GFP）的选择盒。当靶向多顺反子操纵子内的基因时，由于可能对下游基因产生极性效应，依赖这种策略可能会变得复杂。本文描述了一种无抗性盒等位基因交换诱变（FLAEM）方法，其开发目的是减轻盒诱导的极性效应。FLAEM利用Cre-loxP基因组编辑在通过等位基因交换进行靶向缺失后去除选择盒。所得菌株包含一个或多个编码序列的无标记基因缺失。这项技术有助于直接评估基因功能，并扩展了沙眼衣原体基因操作工具的种类。
