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首页> 外文期刊>Genomics >Synergy between tetA and rpsL provides high-stringency positive and negative selection in bacterial artificial chromosome vectors.
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Synergy between tetA and rpsL provides high-stringency positive and negative selection in bacterial artificial chromosome vectors.

机译：tetA和rpsL之间的协同作用可在细菌人工染色体载体中提供高严格性的正负选择。

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摘要

Bacterial artificial chromosome (bacmid) vectors are used to stably propagate large, complex fragments of cloned DNA and are a core technology for functional genomics. The simplest method of analyzing bacmid clones would involve a direct mutagenesis or allele exchange protocol utilizing positive and negative selectable markers. The utility of three different negative selectable markers to function in the context of a bacmid vector was therefore investigated: sacB from Bacillus subtilis, which confers sensitivity to sucrose; tetA from TN10, which confers resistance to tetracycline, osmotic sensitivity, and sensitivity to kanamycin and streptomycin; and rpsL from Escherichia coli, which confers sensitivity to streptomycin. When expressed individually in the context of a bacmid vector, each of these markers confers a similar stringency of negative selection, with plating efficiencies on selective media of 2.3 x 10(-5), 9.4 x 10(-4), and 5.7 x 10(-5), respectively. However coexpression of rpsL and tetA results in a synergistic enhancement of the osmotic, kanamycin, and streptomycin sensitivities, with a stringency of selection of approximately 50- to approximately 1000-fold over that obtained with rpsL or tetA alone and approximately 20-fold more than that obtained using sacB. The combination of rpsL and tetA thus serves as the most efficient positive and negative selectable marker system described to date. Copyright 2001 Academic Press.
机译：细菌人工染色体（bacmid）载体用于稳定繁殖克隆的DNA的大而复杂的片段，并且是功能基因组学的核心技术。分析杆状病毒质粒克隆的最简单方法涉及利用正负选择标记进行直接诱变或等位基因交换方案。因此，研究了三种不同的阴性选择标记在杆状病毒载体中起作用的效用：来自枯草芽孢杆菌的sacB，赋予了对蔗糖的敏感性; TN10的tetA，赋予四环素抗性，渗透敏感性以及对卡那霉素和链霉素的敏感性;和来自大肠杆菌的rpsL，赋予对链霉素的敏感性。当在杆状病毒载体中单独表达时，这些标记物中的每一个都赋予相似的否定选择严格性，在选择性培养基上的铺板效率分别为2.3 x 10（-5），9.4 x 10（-4）和5.7 x 10 （-5）。然而，rpsL和tetA的共表达导致渗透，卡那霉素和链霉素敏感性的协同增强，选择严格性比单独使用rpsL或tetA时高约50到约1000倍，比使用sacB获得的结果。 rpsL和tetA的组合因此成为迄今为止描述的最有效的正负选择标记系统。版权所有2001，学术出版社。

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