Nonreplicating intracellular bacterial vector for conjugative DNA transfer into mitochondria.
Nonreplicating intracellular bacterial vector for conjugative DNA transfer into mitochondria.
复制标题
用于将 DNA 接合转移至线粒体的非复制细胞内细菌载体。
DOI：
10.1007/s11095-012-0701-0
复制
发表时间：
2012
影响因子：
3.7
通讯作者：
Koob,MichaelD
中科院分区：
文献类型：
--
作者：
Yoon,YoungGeol;Koob,MichaelD
目的我们以前已经证明，DNA构建物可以通过接合转移的过程从大肠杆菌宿主导入到分离的线粒体中。为了将DNA转移到细胞中的线粒体，我们着手产生一种能够将自己引入哺乳动物细胞细胞质的接合大肠杆菌菌株。方法我们现在已经开发了一种制造E的方法。能产生非复制子细胞群体的肠系膜。我们利用这种方法对兼性细胞内侵袭性大肠杆菌(EIEC)进行了改造，并将接合功能引入到该新菌株中。结果我们证明该新菌株能够产生大量的非复制细胞群，这些细胞能够与其他细胞接合转移，并且可以很容易地入侵哺乳动物组织培养细胞，并在细胞质中存活数天，并且不会杀死入侵的哺乳动物细胞。结论我们成功地构建了一株适合细胞内接合转移的大肠杆菌宿主，但由于缺乏合适的线粒体筛选或可选择的标记，我们尚不能确定这些细菌载体是否真的能够将DNA转移到细胞内线粒体。
PurposeWe have previously shown that DNA constructs can be introduced into isolated mitochondria through the process of conjugative transfer from an E. coli host. We set out to generate a conjugative E. coli strain that would be able to introduce itself into the cytoplasm of a mammalian cell for the purpose of transferring DNA into the mitochondria in the cell.MethodsWe have now developed a method for makingE. colistrains from which nonreplicating populations of daughter cells can be generated. We used this approach to modify a facultative intracellular enteroinvasive E. coli (EIEC) and introduced conjugative functions to this new strain.ResultsWe demonstrate that this new strain can generate large populations of nonreplicating cells that are capable of conjugative transfer to other cells and can readily invade mammalian tissue culture cells, live in the cytoplasm of the cell for several days, and that do not kill the invaded mammalian cell.ConclusionsWe successfully constructed an E. coli host suitable for intracellular conjugative transfer but, due to the lack of suitable mitochondrial screening or selectable markers, we have not yet been able to determine if these bacterial vectors can in fact transfer DNA into intracelluar mitochondria.