Nuclear Entry of DNA and Transgene Expression in Dividing and Non-dividing Cells.
Nuclear Entry of DNA and Transgene Expression in Dividing and Non-dividing Cells.
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分裂和非分裂细胞中 DNA 的核进入和转基因表达。
DOI：
10.1007/s12195-023-00784-w
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发表时间：
2023
影响因子：
2.8
通讯作者：
Yuan,Fan
中科院分区：
文献类型：
--
作者：
Sylvers,Justin;Wang,Yifei;Yuan,Fan
在哺乳动物细胞中，质粒DNA （pDNA）必须被传递到细胞核中进行转基因表达。这种进入可能在分裂细胞中核膜破裂和重组时被动发生，也可能通过核孔复合物主动发生。本研究的目的是探讨这两种途径对pDNA核进入和随后的基因表达的相对重要性。方法为了测量转染细胞中编码增强绿色荧光蛋白（EGFP）的pDNA的细胞核进入，我们开发了一种基于单分子水平pDNA检测的杂交探针和自动图像分析的细胞核中pDNA定量分析的灵敏技术。在配对实验中，我们使用mRNA靶向杂交探针来量化每个细胞的报告基因mRNA表达，并使用流式细胞术来量化EGFP的表达。结果发现pDNA在细胞核中的分布有点状和弥漫性两种模式，分别在阻滞细胞和未阻滞细胞中占优势。细胞周期阻滞使弥漫性pDNA减少，点状pDNA增加。其净效应是核内pDNA总量的减少。此外，细胞周期阻滞增加了报告基因mRNA的合成，但对报告蛋白的表达没有实质性影响。结论pDNA在细胞分裂过程中进入细胞核并不一定会导致高水平的转基因表达。他们还表明，点状pDNA比细胞核中的弥漫性pDNA更具转录活性。这些数据将有助于进一步研究非病毒基因传递的机制。
IntroductionPlasmid DNA (pDNA) must be delivered into the nucleus for transgene expression in mammalian cells. The entry may happen passively during the nuclear envelope breakdown and reformation in dividing cells or actively through the nuclear pore complexes. The goal of this study was to investigate the relative importance of these two pathways for pDNA nuclear entry and subsequent gene expression.MethodsTo measure nuclear entry of pDNA encoding enhanced green florescence protein (EGFP) in electrotransfected cells, we developed a sensitive technique for quantitative analysis of pDNA in the nuclei, based on a hybridization probe for pDNA detection at the single molecule level and automatic image analysis. In matched experiments, we used an mRNA targeted hybridization probe to quantify reporter mRNA expression per cell, and flow cytometry to quantify expression of EGFP.ResultsWe discovered two distinct patterns of pDNA distribution in the nuclei: punctate and diffuse, which were dominant in arrested and unarrested cells, respectively. The cell cycle arrest decreased diffuse pDNA and increased punctate pDNA. Its net effect was a decrease in the total intranuclear pDNA. Additionally, the cell cycle arrest increased the reporter mRNA synthesis but had no substantial impact on reporter protein expression.ConclusionResults from the study demonstrated that the efficient nuclear entry of pDNA during cell division did not necessarily lead to a high level of transgene expression. They also suggested that the punctate pDNA was more transcriptionally active than diffuse pDNA in the nuclei. These data will be useful in future studies for understanding mechanisms of nonviral gene delivery.