Mycobacterial recombineering.
Mycobacterial recombineering.
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DOI：
10.1007/978-1-4939-2450-9_10
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发表时间：
2015-01-01
期刊：
影响因子：
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通讯作者：
Sassetti, Christopher M
中科院分区：
文献类型：
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作者：
Murphy, Kenan C;Papavinasasundaram, Kadamba;Sassetti, Christopher M
精确敲除或修饰结核分枝杆菌基因对于鉴定这种重要细菌的生长和致病性的重要功能至关重要。先前已经描述了使用非复制载体和转导颗粒将基因敲除底物引入结核分枝杆菌的方案，其中宿主的内源性重组系统（同源和非同源）竞争将修饰的等位基因转移到染色体上。过去16年来首次在实验室和大肠杆菌致病性菌株中引入的重组技术已被开发用于结核分枝杆菌。本章描述的是分枝杆菌Che9c噬菌体RecET重组系统的使用，该系统已被用于在结核分枝杆菌和耻毛分枝杆菌染色体中以非常高的频率进行基因敲除、报告基因融合、启动子替换和单碱基对修饰。与先前描述的结核分枝杆菌基因敲除方案相比，重组通常可以在更短的时间内观察到更高的成功率。
The precise knockout or modification of Mycobacterium tuberculosis genes has been critical for the identification of functions important for the growth and pathogenicity of this important bacterium. Schemes have been previously described, using both non-replicating vectors and transducing particles, for the introduction of gene knockout substrates into M. tuberculosis, where the endogenous recombination systems of the host (both homologous and illegitimate) compete for transfer of the modified allele to the chromosome. Recombineering technologies, first introduced in laboratory and pathogenic strains of Escherichia coli over the last 16 years, have been developed for use in M. tuberculosis. Described in this chapter is the use of the mycobacterial Che9c phage RecET recombination system, which has been used to make gene knockouts, reporter fusions, promoter replacements, and single base pair modifications within the M. tuberculosis and M. smegmatis chromosomes at very high frequency. Higher success rates, in a shorter period of time, are routinely observed when recombineering is compared to previously described M. tuberculosis gene knockout protocols.