Postmitotic nuclear retention of episomal plasmids is altered by DNA labeling and detection methods
Postmitotic nuclear retention of episomal plasmids is altered by DNA labeling and detection methods
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DOI：
10.1016/j.ymthe.2005.05.001
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发表时间：
2005-09-01
影响因子：
12.4
通讯作者：
Dean, DA
中科院分区：
文献类型：
--
作者：
Gasiorowski, JZ;Dean, DA
非病毒基因治疗的一个经常被忽视的方面是质粒在转染细胞内的维持和定位。在这项研究中，我们量化了单核细胞分裂后微注射细胞内质粒的核保留。我们使用了几种市售试剂，用荧光团标记质粒，用于我们的显微注射跟踪实验。有趣的是，用不同的技术标记的质粒产生了截然不同的结果。裸质粒直接注射到细胞核中，然后通过原位杂交检测到，在有丝分裂后几乎只在子细胞的细胞核中发现，并且在子细胞核之间以正态高斯分布分布。用荧光肽核酸标记的质粒获得了相同的结果。然而，当用几种市售的荧光DNA标记试剂盒标记质粒时，将荧光团随机附着在整个质粒上并注射到HeLa细胞核中，细胞分裂后将修饰的质粒从子核中排除。综上所述，这些结果表明，裸质粒在细胞分裂后保留在细胞核中，并可能继续在子细胞中表达。我们的研究结果表明，标记技术本身可以对质粒运输产生显著的改变。
One often overlooked aspect of nonviral gene therapy is the maintenance and localization of plasmids within a transfected cell. In this study we have quantified the nuclear retention of plasmids within microinjected cells after a single round of cell division. We employed several commercially available reagents to label plasmids with fluorophores for our microinjection tracking experiments. Interestingly, plasmids labeled with different techniques produced drastically different results. Naked plasmids microinjected directly into nuclei and later detected by in situ hybridization were found almost exclusively within the nuclei of the daughter cells after mitosis and were partitioned between the daughter nuclei with a normal, Gaussian distribution. Identical results were obtained with plasmids labeled with a fluorescent peptide nucleic acid. However, when plasmids were labeled with several commercially available fluorescent DNA labeling kits that randomly attach fluorophores to the entire plasmid and injected into HeLa cell nuclei, the modified plasmids were excluded from daughter nuclei after cell division. Taken together, these results suggest that naked, unmodified plasmids are retained in the nucleus following cell division and likely continue to express in the daughter cells. Our results demonstrate the significant alterations in episome localization that the labeling technique itself can have on plasmid trafficking.