A novel antibiotic free plasmid selection system: Advances in safe and efficient DNA therapy
A novel antibiotic free plasmid selection system: Advances in safe and efficient DNA therapy
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DOI：
10.1002/biot.200700141
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发表时间：
2008-01-01
影响因子：
4.7
通讯作者：
Grobherr, Reingard
中科院分区：
文献类型：
--
作者：
Mairhofer, Jurgen;Pfaffenzeller, Irene;Grobherr, Reingard
抗生素抗性基因在递送质粒中的存在是现代基因治疗和DNA疫苗应用的缺点之一。在这里，我们描述了一种策略，允许在细菌宿主中进行质粒选择，而不需要任何选择标记。对几种细菌菌株进行了改造，使质粒的复制抑制因子RNA I能够通过RNA-RNA反义反应抑制生长必需基因的翻译。对必需基因（murA）进行修饰，使阻遏蛋白（tetR）阻碍其表达。只有在质粒和RNA 1存在的情况下，tetR才被下调，murA才被表达。不同的市售质粒可以通过各种修饰的大肠杆菌菌株来选择。我们进一步设计了一个不含任何选择标记的极简质粒。当使用修饰的菌株JM 109-murselect进行选择时，检查的所有克隆（n=6）都含有质粒。因此，我们已经设计了细菌宿主菌株，其首次用于选择和维持质粒，而不使用质粒上的任何选择标记或其它附加序列。因此，这样的质粒可能不仅更安全，而且由于其减小的尺寸，预期对制造商有利并且转染效率更高。
The presence of antibiotic resistance genes in the delivered plasmids is one of the drawbacks of modern gene therapy and DNA vaccine applications. Here, we describe a strategy that allows for plasmid selection in bacterial hosts, without the requirement of any selection marker. Several bacterial strains were modified, so that the plasmid's replicational inhibitor RNA I could suppress the translation of a growth essential gene by RNA-RNA antisense reaction. An essential gene (murA) was modified such that a repressor protein (tetR) would hamper its expression. Only in the presence of plasmid and, hence, RNA 1, was tetR turned down and murA expressed. Different commercially available plasmids could be selected by various modified Escherichia coli strains. We further designed a minimalistic plasmid devoid of any selection marker. All of the clones (n=6) examined, when the modified strain JM109-murselect was used for selection, contained plasmids. Thus, we have designed bacterial host strains that for the first time serve to select and maintain plasmids without the use of any selection marker or other additional sequence on the plasmid. Consequently, such plasmids may not only be safer, but due to their decreased size, advantages for the manufacturer and higher transfection efficiencies are anticipated.