Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.
Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.
Bioelectrochemistry. 2023 Oct;153:108491. doi: 10.1016/j.bioelechem.2023.108491. Epub 2023 Jun 20.
Electrotransfection is a non-viral method for delivery of nucleic acids into cells. In our previous study, we have determined the minimal copy number of plasmid DNA (pDNA) per cell required for transgene expression post electrotransfection, and developed a statistical framework to predict the pDNA copy number in the nucleus. To experimentally verify the prediction, the current study was designed to quantify the average copy number of pDNA per nucleus post electrotransfection. To achieve it, we developed a novel approach to effectively obtain isolated nuclei with minimal contamination by extranuclear pDNA. This sample preparation method enabled us to accurately measure intranuclear pDNA using quantitative real-time PCR. The data showed that the copy number of pDNA per nucleus was dependent on the period of cell culture post pulsing and the pDNA dose for electrotransfection. Additionally, the data were used to improve the statistical framework for understanding kinetics of pDNA transport in cells, and predicting how the kinetics depended on different factors. It is expected that the framework and the methodology developed in the current study will be useful for evaluating factors that may affect kinetics and mechanisms of pDNA transport in cells.
电穿孔是一种将核酸递送入细胞的非病毒方法。在我们之前的研究中，我们已经确定了电穿孔后转染基因表达所需的质粒 DNA（pDNA）的最小细胞内拷贝数，并开发了一种统计框架来预测细胞核内的 pDNA 拷贝数。为了实验验证该预测，本研究旨在定量测定电穿孔后每个核内的 pDNA 的平均拷贝数。为了实现这一目标，我们开发了一种新的方法，可有效获得核内无明显核外 pDNA 污染的分离核。这种样品制备方法使我们能够使用定量实时 PCR 准确测量核内 pDNA。数据表明，每个核内的 pDNA 拷贝数取决于脉冲后细胞培养的时间以及电穿孔的 pDNA 剂量。此外，这些数据还可用于改进统计框架，以了解 pDNA 在细胞内转运的动力学，并预测动力学如何取决于不同的因素。预计本研究中开发的框架和方法将有助于评估可能影响 pDNA 在细胞内转运动力学和机制的因素。
