Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis.
Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis.
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DOI：
10.3791/60848
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发表时间：
2020-01-30
期刊：
影响因子：
--
通讯作者：
Fields KA
中科院分区：
文献类型：
--
作者：
Keb G;Fields KA
沙眼衣原体是一种专性细胞内病原体，历史上一直难以基因操纵。由于缺乏遗传工具，在阐明沙眼衣原体用于创建和维持细胞内特权生态位的机制方面的明确进展受到限制。幸运的是，最近在基因操作技术方面有了一些新的进展。其中之一是荧光报道的等位基因交换诱变（FRAEM）的发展。该方法允许靶向基因缺失，并插入编码抗生素耐药性和绿色荧光蛋白（GFP）的选择盒。由于对下游基因的潜在极性效应，当靶向多反反子操纵子内的基因时，依赖这种策略可能会变得复杂。Floxed盒式等位基因交换诱变（FLAEM）是为了减轻盒式诱导的极性效应而开发的。FLAEM利用Cre-loxP基因组编辑，通过等位基因交换去除靶向缺失后的选择盒。所得到的菌株包含一个或多个编码序列的无标记基因缺失。这项技术促进了基因功能的直接评估，并扩大了沙眼衣原体基因操作的工具库。本文描述了一种利用柔丝盒等位基因交换诱变（flem）对沙眼衣原体进行靶向、无标记基因缺失的方法。
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. Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.
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影响因子：
3.1
作者：
Song, Lihua;Carlson, John H.;Caldwell, Harlan D.
通讯作者：
Caldwell, Harlan D.
DOI：
10.1073/pnas.92.11.4877
发表时间：
1995-05-23
影响因子：
11.1
作者：
HACKSTADT, T;SCIDMORE, MA;ROCKEY, DD
通讯作者：
ROCKEY, DD
影响因子：
3.2
作者：
Keb, G.;Hayman, R.;Fields, K. A.
通讯作者：
Fields, K. A.
影响因子：
4.4
作者：
Seth-Smith, Helena M. B.;Harris, Simon R.;Clarke, Ian N.
通讯作者：
Clarke, Ian N.
影响因子：
3.7
作者：
Johnson CM;Fisher DJ
通讯作者：
Fisher DJ